Synthetic Surfaces for Differentiating Stem Cells into Cardiomyocytes

ABSTRACT

Synthetic surfaces capable of supporting culture of eukaryotic cells including stem cells and undifferentiated human embryonic stem cells in a chemically defined medium include a swellable (meth)acrylate layer and a polypeptide conjugated to the swellable (meth)acrylate layer. The swellable (meth)acrylate layer may be formed by polymerizing monomers in a composition that includes a carboxyl group-containing (meth)acrylate monomer, a cross-linking (di- or higher-functional) (meth)acrylate monomer, and a hydrophilic monomer capable of polymerizing with the carboxyl group-containing (meth)acrylate monomer and the cross-linking (meth)acrylate monomer. The swellable (meth)acrylate layer has an equilibrium water content in water of between about 5% and about 70%. The conjugated peptide may include an RGD amino acid sequence.

RELATED APPLICATION(S)

This application claims the benefit of U.S. Provisional Application Ser.No. 61/062,890 filed Jan. 30, 2008 and entitled “Synthetic Surfaces forCulturing Undifferentiated Stem Cells in Chemically Defined Media” andU.S. Provisional Application Ser. No. 61/062,937 filed Jan. 30, 2008 andentitled “Stem Cell Article and Screening.”

FIELD

The present disclosure relates to cell culture articles, and moreparticularly to articles for supporting the culture of undifferentiatedstem cells in a chemically defined medium.

BACKGROUND

Pluripotent stem cells such as human embryonic stem cells (hESCs) havethe ability to differentiate into any of the three germ layers, givingrise to any adult cell type in the human body. This unique propertyprovides a potential for developing new treatments for a number ofserious cell degenerative diseases, such as diabetes, spinal chordinjury, heart diseases and the like. However there remain obstacles inthe development of such hESC-based treatments. Such obstacles includeobtaining and maintaining adequate numbers of undifferentiated hESCs incell and tissue culture and controlling their differentiation in orderto produce specific cell types. Stem Cell Cultures, such as hESC cellcultures are typically seeded with a small number of cells from a cellbank or stock and then amplified in the undifferentiated state untildifferentiation is desired for a given therapeutic application. Toaccomplish this, the hESC or their differentiated cell populations arecurrently cultured in the presence of surfaces or media containinganimal-derived components, such as feeder layers, fetal bovine serum, orMATRIGEL™. These animal-derived additions to the culture environmentexpose the cells to potentially harmful viruses or other infectiousagents which could be transferred to patients or compromise generalculture and maintenance of the hESCs. In addition, such biologicalproducts are vulnerable to batch variation, immune response and limitedshelf-life.

While undifferentiated stem cells have been grown in chemically definedmedia on animal-derived surfaces such as MATRIGEL™ and proteins such asserum proteins or extra-cellular matrix proteins, to date, a completelyanimal product free system employing a chemically defined medium and asynthetic non-protein surface has not been identified for long-termculturing of undifferentiated stem cells.

BRIEF SUMMARY

The present disclosure describes synthetic surfaces that may be usefulin the culture of eukaryotic cells including stem cells andundifferentiated stem cells in chemically defined media.Undifferentiated stem cells may remain undifferentiated for 5, 7, 10 ormore passages when cultured on the synthetic substrates.

In various embodiments, a cell culture article includes a substratehaving a surface. A swellable (meth)acrylate layer is disposed on thesurface of the substrate. The swellable (meth)acrylate layer is formedfrom a composition that includes a carboxyl group-containing(meth)acrylate monomer, a cross-linking (di- or higher-functional)(meth)acrylate monomer, and a hydrophilic monomer capable ofpolymerizing with the carboxyl group-containing (meth)acrylate monomerand the cross-linking (meth)acrylate monomer. The swellable(meth)acrylate layer has an equilibrium water content in water ofbetween about 5% and about 70%. A peptide is conjugated to the swellable(meth)acrylate layer. The peptide contains an RGD amino acid sequence.The cell culture article may support culture and maintenance ofundifferentiated stem cells, such as human embryonic stem cells, in achemically defined medium.

In various embodiments, a method for producing a cell culture articleincludes disposing monomers on a substrate surface of the cell culturearticle. The monomers disposed on the surface include a carboxylgroup-containing (meth)acrylate monomer, a cross-linking (meth)acrylatemonomer, and a hydrophilic monomer capable of polymerizing with thecarboxyl group-containing (meth)acrylate monomer and the cross-linking(meth)acrylate monomer. The method further includes polymerizing themonomers on the substrate surface to form a swellable (meth)acrylatelayer having an equilibrium water content in water of between about 5%and about 70%. The method also includes conjugating to the swellable(meth)acrylate layer a peptide which may be an RGD-containingpolypeptide.

One or more of the various embodiments presented herein provide one ormore advantages over prior surfaces for culturing stem cells,particularly undifferentiated stem cells. For example, the syntheticsurfaces reduce potential contamination issues associated with surfaceshaving components obtained from or derived from animal sources. Suchsurfaces may also provide for improved shelf life compared to thosesurfaces with biological components. The ability to cultureundifferentiated stem cells in chemically-defined media further reducespotential contamination issues. In addition, there will likely be lessbatch to batch variation in the performance capability of the syntheticsurfaces or chemically defined media, resulting in improvedreproducibility of culture results and expectations. These and otheradvantages will be readily understood from the following detaileddescriptions when read in conjunction with the accompanying drawings

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B are schematic diagrams of side views of synthetic swellable(meth)acrylate layer coated articles.

FIG. 2A-C are schematic diagrams of cross sections of cell culturearticles having a well. Uncoated (2A); coated surface (2B); and coatedsurface and side walls (2C).

FIG. 3A is an image of fluorescence intensity of polypeptide conjugatedto 1 micrometer thick layers of swellable (meth)acrylate coating in amulti-well plate.

FIG. 3B is an image of fluorescence intensity of polypeptide conjugatedto 0.1 micrometer thick layers of swellable (meth)acrylate coating in amulti-well plate.

FIG. 3C is a graph of polypeptide concentration vs. fluorescenceintensity for polypeptides coated on 1 micrometer or 0.1 micrometerthick surfaces.

FIG. 4 is a graph of polypeptide concentration vs. absorbance at 570 nmfor data obtained from a QuantiPro BCA assay.

FIG. 5 is a graph of polypeptide concentration vs. absorbance at 570 nmnormalized to laminin, showing a results from an assay designed todetermine the adherence of HT-1080 cells to various substrates.

FIG. 6 is a graph illustrating the relative number of undifferentiatedhESC (by AttoPhos fluorescence intensity) cultured in a chemicallydefined medium on swellable (meth)acrylate substrates having particularconjugated polypeptides.

FIGS. 7A-C are images of colonies of undifferentiated human embryonicstem cells cultured in a chemically defined medium on a MATRIGEL™substrate (A), a swellable (meth)acrylate with conjugated peptide (SEQID NO:4) (as identified in FIG. 6) (B), or swellable (meth)acrylatealone (C).

FIG. 8 is a graph illustrating the relative number of undifferentiatedhuman embryonic stem cells (by AttoPhos fluorescence intensity) culturedin a chemically defined medium on a swellable (meth)acrylate conjugatedwith varying concentrations of peptide (SEQ ID NO:4) (as identified inFIG. 6).

FIG. 9 is a graph illustrating the relative number of undifferentiatedhuman embryonic stem cells (by AttoPhos fluorescence intensity) culturedin a chemically defined medium cultured on embodiments of swellable(meth)acrylate substrates.

FIGS. 10A and 10B are bar graphs illustrating the relative number ofundifferentiated H7 hES cells (by AttoPhos fluorescence intensity) underchemically defined medium condition on embodiments of swellable(meth)acrylate formulations after conjugated with peptideAc-KGGNGEPRGDTYRAY-NH₂ (SEQ ID NO:4).

FIG. 11 is a bar graph illustrating the relative number ofundifferentiated H7 hES cells (by AttoPhos fluorescence intensity)cultured on 10-, 100- and 1000-concentrations of 1-FN (SEQ ID NO:12),s-FN (SEQ ID NO:11) and Ac-KGGNGEPRGDTYRAY-NH₂ (SEQ ID NO:4) conjugatedto embodiments of swellable (meth)acrylate coatings.

FIG. 12 is a bar graph showing relative HT-1080 attachment (via OD 570nm) to di-sulfide bonded, cyclized polypeptides conjugated to aswellable (meth)acrylate layer.

FIG. 13 is a bar graph showing relative HT-1080 attachment (via OD 570nm) to amide bonded, cyclized polypeptides conjugated to a swellable(meth)acrylate layer.

FIG. 14 is a bar graph illustrating the relative number ofundifferentiated H7 hES cells (by AttoPhos fluorescence intensity)cultured on a swellable (meth)acrylate layer with conjugated amidebonded, cyclized polypeptides.

FIG. 15 is a bar graph illustrating the relative number ofundifferentiated H7 hES cells (by AttoPhos fluorescence intensity)cultured on a swellable (meth)acrylate layer with conjugated di-sulfidebonded, cyclized polypeptides.

FIG. 16 is a graph showing the doubling time for undifferentiated H7hESCs cultured under chemically-defined, animal produce free mediumconditions on a MATRIGEL™ (MG) surface, a swellable (meth)acrylate layerwith conjugated defined RGD-containing peptide (SEQ ID NO:4) (BSP), anda swellable (meth)acrylate layer with conjugated defined RGD-containingpeptide with a linker (BSP-PE04).

FIGS. 17A-C are images comparing the survival/proliferation of humanmesenchymal stem cells (hMSC) on SAP-BSP peptide-acrylate with standardTCT plastic cell culture ware on day 7 post-seeding.

FIG. 18 is a bar graph showing a comparison of hMSC cell number on TCTand SAP-BSP after 7 days in culture under different media conditionsincluding defined media.

FIG. 19 is a graph showing a comparison of the H7 hESCs doubling timewhen cultured on an embodiment of the cell culture surface of thepresent invention compared to MATRIGEL™ coating across 10 passages.

FIG. 20 shows the results of flow cytometry analyses of hESC-specificmarkers for H7 hESC cultured on MATRIGEL™ control or SAP-BSP surface for10 passages.

FIG. 21A is a fluorescence image showing actinin and Nkx2.5+ stainingfor cardiomyocytes differentiated on SAP-BSP peptide conjugated acrylatesurfaces. FIG. 21B shows flow cytometry data for αActinin and FIG. 21Cshows flow cytometry data for Nkx2.5 cardiomyocytes differentiated fromH7 hESC on an embodiment of the cell culture surface of the presentinvention.

FIG. 22 is a bar graph comparing HT1080 cell proliferation (viaabsorbance at 490 nm) data of HT-108-cells on TCT, SAP-BSP surface, inthe presence and absence of serum.

FIG. 23 are images comparing the morphology of HT-1080 cells on TCT and(meth)acrylate coating and BSP conjugated coatings in the presence andabsence of serum.

FIG. 24 is a bar graph comparison of the estimated number of viablecells (absorbance of LDH activity) on SA-Ps, Collagen I and CellBind®treated Topas™.

The drawings depicted in FIGS. 1-2 are not necessarily to scale. Likenumbers used in the FIGS. 1-2 refer to like components. However, it willbe understood that the use of a number to refer to a component in agiven figure is not intended to limit the component in the other figurelabeled with the same number. In addition, the use of different numbersto refer to components is not intended to indicate that the differentnumbered components cannot be the same or similar.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings that form a part hereof, and in which are shown byway of illustration several specific embodiments of devices, systems andmethods. It is to be understood that other embodiments are contemplatedand may be made without departing from the scope or spirit of thepresent disclosure. The following detailed description, therefore, isnot to be taken in a limiting sense.

All scientific and technical terms used herein have meanings commonlyused in the art unless otherwise specified. The definitions providedherein are to facilitate understanding of certain terms used frequentlyherein and are not meant to limit the scope of the present disclosure.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” encompass embodiments having pluralreferents, unless the content clearly dictates otherwise. As used inthis specification and the appended claims, the term “or” is generallyemployed in its sense including “and/or” unless the content clearlydictates otherwise.

As used herein, “providing” an article in the context of a method meansproducing, purchasing, fabricating, supplying or otherwise obtaining thearticle so that the article may be used in the method.

Peptide sequences are referred to herein by their one letter and bytheir three letter codes. These codes may be used interchangeably.

As used herein, “monomer” means a compound capable of polymerizing withanother monomer, (regardless of whether the “monomer” is of the same ordifferent compound than the other monomer), which compound has amolecular weight of less that about 1000 Dalton. In many cases, monomerswill have a molecular weight of less than about 400 Dalton.

As used herein, “synthetic coating” or “synthetic polymeric layer” meansa layer of polymer material disposed on a cell culture substrate. Thepolymeric coating does not include animal-derived materials oringredients. For example, a synthetic coating does not have proteinsisolated from an animal source. In embodiments, swellable (meth)acrylatelayers (SA layers) as described herein provide a synthetic coating. Inembodiments, a suitable adhesion polypeptide or combinations ofpolypeptides may be conjugated to the synthetic coating layer formingswellable(meth)acrylate-peptide conjugated layers (SAP layers).

As used herein “peptide” and “polypeptide” mean a sequence of aminoacids that may be chemically synthesized or may be recombinantlyderived, but that are not isolated as entire proteins from animalsources. For the purposes of this disclosure, peptides and polypeptidesare not whole proteins. Peptides and polypeptides may include amino acidsequences that are fragments of proteins. For example peptides andpolypeptides may include sequences known as cell adhesion sequences suchas RGD. In some embodiments, the peptides of the present invention arebetween three and 30 amino acids in length. In some embodiments, thepeptides of the present invention are 15mers, 12mers, 10mers or thelike. In embodiments, peptides may include linker sequences such as KGG(LysGlyGly) or KYG (LysTyrGly) which provide a Lys residue which mayprovide a functional group for conjugation of the peptide. Peptides maybe acetylated (e.g. Ac-LysGlyGly) or amidated (e.g. SerLysSer-NH₂) toprotect them from being broken down by, for example, exopeptidases. Itwill be understood that these modifications are contemplated when asequence is disclosed. In additional embodiments, peptides may beconjugated to spacer moieties such as PEO. PEO may be present as, forexample, PEO₄ or PEO₁₂ or any length spacer.

As used herein, a “(meth)acrylate monomer” means a compound having atleast one ethylenically unsaturated moiety (an acrylate moiety or amethacrylate moiety). “Poly(meth)acrylate”, as used herein, means apolymer formed from one or more monomers including at least one(meth)acrylate monomer.

The term “hydrogel” has been used to describe cell culture surfaces.“Hydrogel” has been variously defined to include a gel or gelatin thatcan absorb water in an amount greater than or equal to 30% or up to10,000% of its dry weight. When contacted with water, hydrogels swellbut do not dissolve. The term “hydrogel” is a very broad term,describing a wide range of materials, having a wide range of waterswelling and water absorbing characteristics.

As used herein, “swellable (meth)acrylate” or “SA” means a polymermatrix made from at least one ethylenically unsaturated monomer(acrylate or methacrylate monomers) having at least some degree of crosslinking, and also having water absorbing or water swellingcharacteristics. Swellable (meth)acrylates may be synthetic. That is,they do not contain ingredients that are derived from animals or animalextracts. Swellable (meth)acrylates may be conjugated to polypeptides orproteins (“swellable (meth)acrylate-polypeptide”, “SA-polypeptide” or“SAP”). Polypeptides or peptides are fragments of proteins and may besynthesized, making them synthetic, non-animal-derived materials.Proteins may be isolated from animal-derived material. This SA and SAPmaterial may be referred to as a layer, a coating, a surface, amaterial, or any other term known in the art to refer to a surfacesuitable for cell culture. The particular polypeptide sequence may befurther identified. For example, a SAP surface may be conjugated with aRGD-containing polypeptide and may be identified as SAP-RGD. Or, a SAPsurface may be conjugated to a particular RGD-containing peptide, forexample KGGNGEPRGDTYRAY (SEQ ID NO:4), incorporates the RGD adhesionepitope of Bonesialo protein (BSP) from mouse may be conjugated to aswellable (meth)acrylate polymer (SAP) to form an SAP-BSP coating. Inembodiments of the present disclosure, the term “swellable(meth)acrylate” represents a range of cross-linked acrylate ormethacrylate materials which absorb water, swell in water, and do notdissolve in water. This water-absorbing characteristic can be describedand measured by equilibrium water content (EWC) as shown by Formula 1:

EWC (%)=[(Wgel−Wdry)/(Wgel)]*100.  Formula 1

As used herein, “have”, “having”, “include”, “including”, “comprise”,“comprising” or the like are used in their open ended sense, andgenerally mean “including, but not limited to”. It will be understoodthat “consisting essentially of”, “consisting of”, and the like aresubsumed in “comprising” and the like. Accordingly, a SA surface formedfrom a mixture of monomers comprising a hydrophilic monomer, across-linking monomer and a carboxyl group-containing monomer may beformed from a mixture consisting essentially of, or consisting of, ahydrophilic monomer, a cross-linking monomer and a carboxylgroup-containing monomer.

The present disclosure describes, inter alia, articles having syntheticsurfaces for culturing undifferentiated stem cells. The surfaces maysupport proliferation and maintenance of undifferentiated stem cells inchemically defined media.

1. Cell Culture Article

Referring to FIG. 1A, a schematic diagram of a side view of an article100 for culturing cells is shown. The article 100 includes a basematerial substrate 10 having a surface 15. A swellable (meth)acrylatecoating layer 20 is disposed on the surface 15 of the substrate or basematerial 10. While not shown, it will be understood that swellable(meth)acrylate coating 20 may be disposed on a portion of substrate orbase material 10. The substrate or base material 10 may be any materialsuitable for culturing cells, including a ceramic substance, a glass, aplastic, a polymer or co-polymer, any combinations thereof, or a coatingof one material on another. Such substrate or base materials 10 includeglass materials such as soda-lime glass, pyrex glass, vycor glass,quartz glass; silicon; plastics or polymers, including dendriticpolymers, such as poly(vinyl chloride), poly(vinyl alcohol), poly(methylmethacrylate), poly(vinyl acetate-co-maleic anhydride),poly(dimethylsiloxane) monomethacrylate, cyclic olefin polymers,fluorocarbon polymers, polystyrenes, polypropylene, polyethyleneimine;copolymers such as poly(vinyl acetate-co-maleic anhydride),poly(styrene-co-maleic anhydride), poly(ethylene-co-acrylic acid) orderivatives of these or the like.

As used herein, “cyclic olefin copolymer” means a polymer formed frommore than one monomer species, where at least one of the monomer speciesis a cyclic olefin monomer and at least one other monomer species is nota cyclic olefin monomer species. In many embodiments, cyclic olefincopolymers are formed from ethylene and norbonene monomers. Cyclicolefin copolymer resins are commercially available with trade name ofTOPAS® from Boedeker Plastics, Inc.

Examples of articles 100 suitable for cell culture include single andmulti-well plates, such as 6, 12, 96, 384, and 1536 well plates, jars,petri dishes, flasks, multi-layered flasks, beakers, plates, rollerbottles, slides, such as chambered and multichambered culture slides,tubes, cover slips, bags, membranes, hollow fibers, beads andmicrocarriers, cups, spinner bottles, perfusion chambers, bioreactors,CellSTACK® and fermenters.

Swellable (meth)acrylate (SA) coating 20 provides a surface 25 on whichone or more polypeptides 70 may be conjugated. Of course, the one ormore polypeptides 70 may also be conjugated to the SA layer 20 atlocations beneath the surface 25. As used herein, “conjugated” meanscovalently bound. Covalent binding of polypeptide 70 to SA layer 20 mayoccur through a linker. The polypeptide 70 may be cyclic or linear, ormay contain portions that are cyclic and portions that are linear.

For the purposes of this disclosure “peptide” and “polypeptide” can beused interchangeably. Any suitable SA coating 20 may be applied to orformed on the surface 15 of the substrate 10. In various embodiments,the SA coating comprises, consists essentially of, or consists of,reaction products of one or more hydrophilic (meth)acrylate monomer, oneor more di- or higher-functional (meth)acrylate monomer (“cross-linking”(meth)acrylate monomer), and one or more carboxyl group-containingmonomers. Any suitable hydrophilic (meth)acrylate monomer may beemployed. Examples of suitable hydrophilic (meth)acryate monomersinclude 2-hydroxyethyl methacrylate, di(ethylene glycol)ethyl ethermethacrylate, ethylene glycol methyl ether methacrylate, and the like.In various embodiments, hydrophilic monomers other than (meth)acrylatesmay be used to form the SA coating. These other hydrophilic monomers maybe included in addition to, or in place of, hydrophilic (meth)acrylatemonomers. Such other hydrophilic monomers should be capable ofundergoing polymerizing with (meth)acrylate monomers in the mixture usedto form the swellable (meth)acrylate layer 20. Examples of otherhydrophilic monomers that may be employed to form the SA coating include1-vinyl-2-pyrrolidone, acrylamide,3-sulfopropyldimethyl-3-methylacrylamideopropyl-ammonium, and the like.Regardless of whether a (meth)acrylate monomer or other monomer isemployed, a hydrophilic monomer, in various embodiments, has asolubility in water of 1 gram or more of monomer in 100 grams of water.Any suitable di- or higher-functional (meth)acrylate monomer, such astetra(ethylene glycol) dimethacrylate or tetra(ethylene glycol)diacrylate, may be employed as a cross-linking monomer. Any suitable(meth)acrylate monomer having a carboxyl functional group available forconjugating with a polypeptide 70 after the monomer is incorporated intothe SA coating 20 by polymerization may be employed. The carboxylfunctional group enables conjugation of a peptide or polypeptide usingNHS/EDC chemistry. Examples of suitable carboxyl group-containing(meth)acrylate include 2-carboxyethyl acrylate and acrylic acid.

In various embodiments, the SA layer 20 is formed from monomerscomprising (by percent volume): hydrophilic (meth)acrylate monomer(˜60-90), carboxyl group-containing (meth)acrylate monomer (˜10-40), andcross-linking (meth)acrylate monomer (˜1-10), respectively. It will beunderstood that the equilibrium water content (EWC) of the SA layer maybe controlled by the monomers chosen to form the SA layer. For example,a higher degree of hydrophilicity and a higher percentage of thehydrophilic monomer should result in a more swellable SA layer with ahigher EWC. However, this may be attenuated by increasing thepercentage, or increasing the functionality, of the cross-linkingmonomer, which should reduce the ability of the SA layer to swell andreduce the EWC.

In various embodiments, the specific monomers employed to form the SAlayer and their respective weight or volume percentages are selectedsuch that the resulting SA layer has an EWC of between about 5% andabout 70%. Due in part to the use of a carboxyl containing monomer inthe SAs of various embodiments described herein, the EWC may be pHdependent. For example, the EWC of particular SAs may be higher inphosphate buffer (pH 7.4) than in distilled, deionized water (pH ˜5). Invarious embodiments, the EWC of an SA layer in distilled, deionizedwater is the EWC (in water) of SAs of the present invention may rangebetween 5% and 70%, between 5% and 60%, between 5% and 50%, between 5and 40%, between 5% and 35%, between 10% and 70%, between 10% and 50%between 10 and 40%, between 5% and 35%, between 10% and 35% or between15% and 35% in water. In further embodiments, after the swellable(meth)acrylates have been conjugated with peptides (SAP), the EWC ofembodiments of SAPs of the present invention may be, for example,between 10-40% in water (data not shown).

In cell culture, prepared surfaces are exposed to an aqueous environmentfor extended periods of time. Surfaces that absorb significant water,surfaces that are highly hydrogel-like, may tend to delaminate from asubstrate when exposed to an aqueous environment. This may be especiallytrue when these materials are exposed to an aqueous environment forextended periods of time, such as for 5 or more days of cell culture.Accordingly, it may be desirable for SA and SAP layers to have lower EWCmeasurements, so that they do not absorb as much water, to reduce thelikelihood of delaminating. For example, SA surfaces having an EWC below40% may be particularly suitable for supporting cells in culture,including human embryonic stem cells.

It will be understood that the conjugation of a polypeptide 70 to an SAlayer 20 may affect the swellability and equilibrium water content (EWC)of the SA layer, generally increasing the EWC. The amount of polypeptideconjugated to SA layers tends to be variable and can change depending onthe thickness of the SA layer. Accordingly, the EWC of a SA-polypeptidelayers prepared in accordance with a standard protocol may be variable.For purposes of reproducibility, it may be desirable to measure the EWCof SA layers prior to conjugation with a polypeptide. With this noted,in some embodiments, after the SAs have been conjugated withpolypeptides (SA-polypeptide), the EWC of embodiments of SA-polypeptidelayers may be between about 10% and about 40% in water.

In various embodiments, the SA layer 20 includes polymerized(meth)acrylate monomers formed from a mixture including hydroxyethylmethacrylate, 2-carboxyethylacrylate, and tetra(ethylene glycol)dimethacrylate. In numerous embodiments, the ratio (by volume) ofhydroxyethyl methacrylate, 2-carboxyethylacrylate, and tetra(ethyleneglycol) dimethacrylate used to form the SA layer 20 is about 80/20/3(v/v/v), respectively. In some embodiments, the SA is formulated usingthe following liquid aliquots of monomers (by volume): hydroxyethylmethacrylate (˜60-90), 2-carboxyethylacrylate (˜10-40), andtetra(ethylene glycol) dimethacrylate (˜1-10), respectively. In numerousembodiments, the SA layer 20 consists essentially of polymerizedhydroxyethyl methacrylate, 2-carboxyethylacrylate, and tetra(ethyleneglycol) dimethacrylate monomers. In various embodiments, the SA layer 20is substantially free of polypeptide crosslinkers.

In numerous embodiments, the SA is formulated using the monomers (byvolume for liquid monomers or weight for solid monomers): Monomer X(80), 2-carboxyethylacrylate (20), and tetra(ethylene glycol)dimethacrylate (3). Monomer X is: Hydroxypropyl methacrylate,2-Hydroxyethyl acrylate, 1-vinyl-2-pyrrolidone, di(ethylene glycol)ethyl ether methacrylate, acrylamide, ethylene glycol methyl ethermethacrylate, or 3-sulfopropyldimethyl-3-methacrylamidopropyl-ammonium.

Any suitable polypeptide 70 may be conjugated to the SA layer 20.Preferably, polypeptide 70 includes an amino acid capable of conjugatingto the SA layer 20; e.g. via the free carboxyl group formed from thecarboxyl group-containing (meth)acrylate monomer. By way of example, anynative or biomimetic amino acid having functionality that enablesnucleophilic addition; e.g. via amide bond formation, may be included inpolypeptide 70 for purposes of conjugating to SA layer 20. Lysine,homolysine, ornithine, diaminoproprionic acid, and diaminobutanoic acidare examples of amino acids having suitable properties for conjugationto a carboxyl group of the SA layer 20. In addition, the N-terminalalpha amine of a polypeptide may be used to conjugate to the carboxylgroup, if the N-terminal amine is not capped. In various embodiments,the amino acid of polypeptide 70 that conjugates with the SA layer 20 isat the carboxy terminal position or the amino terminal position of thepolypeptide 70.

In numerous embodiments, the polypeptide 70, or a portion thereof, hascell adhesive activity; i.e., when the polypeptide 70 is conjugated tothe SA layer, the peptide allows a cell to adhere to the surfacecontaining the SA-P layer. By way of example, the polypeptide 70 mayinclude an amino sequence, or a cell adhesive portion thereof, ofcollagen, keratin, gelatin, fibronectin, vitronectin, laminin, or thelike. In various embodiments, polypeptide 70 includes an amino acidsequence of Yaa₁Xaa_(n)ArgGlyAspXaa_(m)Yaa₁ (SEQ ID NO:1) (RGD) whereXaa and Yaa represent any amino acid, synthetic or naturally occurringand n and m can be any integer including 0, SA layers as describedherein provide a synthetic surface to which any suitable adhesionpolypeptide or combinations of polypeptides may be conjugated, providingan alternative to biological substrates or serum that have unknowncomponents. In current cell culture practice, it is known that some celltypes require the presence of a biological polypeptide or combination ofpeptides on the culture surface for the cells to adhere to the surfaceand be sustainably cultured. For example, HepG2/C3A hepatocyte cells canattach to plastic culture ware in the presence of serum. It is alsoknown that serum can provide polypeptides that can adhere to plasticculture ware to provide a surface to which certain cells can attach.However, biologically-derived substrates and serum contain unknowncomponents. For cells where the particular component or combination ofcomponents (peptides) of serum or biologically-derived substrates thatcause cell attachment are known, those known peptides can be synthesizedand applied to an SA layer as described herein to allow the cells to becultured on a synthetic surface having no or very few components ofunknown origin or composition.

In some embodiments, peptide 70 includes an amino acid sequence ofYaa₁Xaa_(n)ArgGlyAspXaa_(m)Yaa₁ (SEQ ID NO:1), where n is an integer of0 to 4, m is an integer of 0 to 5, 1 is 0 or 1 provided that the peptide70 includes at least one Yaa, each Xaa is independently any native orbiomimetic amino acid, and Yaa is any native or biomimetic amino acid,where either Xaa or Yaa has functionality that enables nucleophilicaddition; e.g. via amide bond formation, to a free carboxyl group of SAsurface. In various embodiments, Xaa or Yaa may be lysine, cysteine,homocysteine, penicillamine, ornithine, diaminoproprionic acid, ordiaminobutanoic acid.

In some embodiments, polypeptide 70 includes an amino acid sequence ofZaaXaa_(n)ArgGlyAspXaa_(m)Baa (SEQ ID NO:2), where n is an integer of 0to 4, m is an integer of 0 to 5, each Xaa is independently any native orbiomimetic amino acid, and Zaa and Baa are each independently any nativeor biomimetic amino acid having covalent bonds formed between atoms oftheir respective side chains to form a cyclic polypeptide or portionthereof. In various embodiments, Zaa and Baa are linked via a disulfidebond. For example, Zaa or Baa may be cysteine, homocysteine orpenicillamine. In some embodiments, Zaa and Baa are linked via an amidebond. For example, one of Zaa and Baa is an amino acid with a side chainhaving a free amino group, such as lysine, ornithine, diaminoproprionicacid, or diaminobutanoic acid, and the other of Zaa and Baa is an aminoacid with a side chain having a free carboxyl group, such as asparticacid, glutamic acid, or homoglutamic acid. In some embodiments, apolypeptide 70 according to SEQ ID NO:2 may be further defined asincluding an amino acid sequence ofYaa₁Xaa_(m)ZaaXaa_(n)ArgGlyAspXaa_(m)Baa Xaa_(m)Yaa₁ (SEQ ID NO:3),where n is an integer of 0 to 4, m is an integer of 0 to 5, 1 is 0 or 1provided that the polypeptide includes at least one Yaa or Xaa, each Yaaand Xaa is independently any native or biomimetic amino acid, and Zaaand Baa are each independently any native or biomimetic amino acidhaving covalent bonds formed between atoms of their respective sidechains to form a cyclic portion of the polypeptide, and Yaa or Xaa isany native or biomimetic amino acid having functionality that enablesnucleophilic addition; e.g. via amide bond formation, to a free carboxylgroup of SA layer. While not intending to be bound by theory, it isbelieved that cyclization of a polypeptide containing an RGD sequencemay help maintain a conformation structure in aqueous environments topreserve biologic function relative to linear counterpart polypeptides,particularly when the polypeptides consist of relatively few aminoacids; e.g. less than 10.

In some embodiments, polypeptide 70 comprises an amino acid sequence ofLysGlyGlyAsnGlyGluProArgGlyAspThrTyrArgAlaTry (SEQ ID NO:4). In someembodiments, polypeptide 70 consists essentially of an amino acidsequence of AsnGlyGluProArgGlyAspThrTyrArgAlaTyr (SEQ ID NO:5). In someembodiments, peptide 70 consists essentially of an amino acid sequenceof LysGlyGlyLys⁴AsnGlyGluProArgGlyAspThrTyrArgAlaTyrAsp¹⁷ (SEQ ID NO:6),where Lys⁴ and Asp¹⁷ together form an amide bond to cyclize a portion ofthe polypeptide. In some embodiments, peptide 70 consists essentially ofan amino acid sequence of LysGlyGlyLys⁴GluProArgGlyAspThrTryArgAsp¹³(SEQ ID NO:7), where Lys⁴ and Asp¹³ together form an amide bond tocyclize a portion of the polypeptide. In some embodiments, polypeptide70 consists essentially of an amino acid sequence ofLysGlyGlyCys⁴AsnGlyGluProArgGlyAspThrTyrArgAlaTyrCys¹⁷ (SEQ ID NO:8),where Cys⁴ and Cys¹⁷ together form a disulfide bond to cyclize a portionof the polypeptide. In some embodiments, polypeptide 70 consistsessentially of an amino acid sequence ofLysGlyGlyCys⁴GluProArgGlyAspThrTryArgCys¹³ (SEQ ID NO:9), where Cys⁴ andCys¹³ together form a disulfide bond to cyclize a portion of thepolypeptide. In various embodiments, peptide 70 consists essentially ofan amino acid sequence of XaaGlyGlyAsnGlyGluProArgGlyAspThrTyrArgAlaTyr(SEQ ID NO:10), wherein Xaa is any amino acid. In numerous embodiments,Xaa of SEQ ID NO:10 is lysine. In some embodiments, polypeptide 70consists essentially of an amino acid sequence of GlyArgGlyAspSerProLys(SEQ ID NO:11).

In some embodiments, polypeptide 70 consists essentially of an aminoacid sequence of LysGlyGlyAlaValThrGlyArgGlyAspSerProAlaSerSer (SEQ IDNO:12).

For any of the polypeptide sequences discussed herein, it will beunderstood that a conservative amino acid may be substituted for aspecifically identified amino acid. A “conservative amino acid”, as usedherein, refers to an amino acid that is functionally similar to a secondamino acid. Such amino acids may be substituted for each other in apolypeptide with a minimal disturbance to the structure or function ofthe polypeptide according to well known techniques. The following fivegroups each contain amino acids that are conservative substitutions forone another: Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine(L), Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y),Tryptophan (W); Sulfur-containing: Methionine (M), Cysteine (C); Basic:Arginine (R), Lysine (K), Histidine (H); Acidic: Aspartic acid (I),Glutamic acid (E), Asparagine (N), Glutamine (Q).

A linker or spacer 80, such as a repeating poly(ethylene glycol) linkeror any other suitable linker, may be used to increase distance frompolypeptide 70 to surface 25 of swellable (meth)acrylate layer 20. Thelinker 80 may be of any suitable length. For example, if the linker is arepeating poly(ethylene glycol) linker, the linker may contain between 2and 10 repeating ethylene glycol units. In some embodiments, the linkeris a repeating poly(ethylene glycol) linker having about 4 repeatingethylene glycol units. All, some, or none of the polypeptides 70 may beconjugated to SA layer 20 via linkers 80. A SA layer chain whichprovides a conjugation group may also act as spacer for conjugatedpeptides. Other potential linkers are polypeptide linkers such aspoly(glycine) or poly(β-alanine).

Polypeptide 70 may be conjugated to the SA layer 20 at any density,preferably at a density suitable to support culture of undifferentiatedstem cells or other cell types. Polypeptide 70 may be conjugated to SAlayer 20 at a density of between about 1 pmol per mm² and about 50 pmolper mm² of surface 25 of SA coating 20, which can be estimated by thearea of surface 15 of base material substrate 10 that is coated inembodiments where surface 15 is uniformly coated by SAP layer 20. Forexample, the polypeptide may be present at a density of greater than 5pmol/mm², greater than 6 pmol/mm², greater than 7 pmol/mm², greater than8 pmol/mm², greater than 9 pmol/mm², greater than 10 pmol/mm², greaterthan 12 pmol/mm², greater than 15 pmol/mm², or greater than 20 pmol/mm²of the surface 25 of the SA coating 20. It will be understood that theamount of polypeptide 70 present can vary depending on the compositionof the SA layer 20, the thickness of the SA layer 20 and the nature ofthe polypeptide 70 itself. As discussed below in the Examples, higherdensities of polypeptide 70 may be better able to support attachment andproliferation of undifferentiated stem cells in a chemically definedmedium.

(Meth)acrylate monomers may be synthesized as known in the art orobtained from a commercial vendor, such as Polysciences, Inc., SigmaAldrich, Inc., and Sartomer, Inc. Polypeptides 70 may be synthesized asknown in the art (or alternatively produced through molecular biologicaltechniques) or obtained from a commercial vendor, such as AmericanPeptide Company, CEM Corporation, or GenScript Corporation. Linkers 80may be synthesized as known in the art or obtained from a commercialvendor, such as discrete polyethylene glycol (dPEG) linkers availablefrom Quanta BioDesign, Ltd.

As shown in FIG. 1B, an intermediate layer 30 may be disposed betweensurface 15 of base material 10 and the SA coating 20. Intermediate layer30 may be configured to improve binding of coating 20 to substrate 10,to facilitate monomer spreading, to render portions of the surface 10that are uncoated cytophobic to encourage cell growth on coated areas,to provide a substrate compatible with a monomer or solvent where themonomer or solvent is incompatible with the base material 10, to providetopographical features if desired through, for example, patternedprinting, or the like. For example, if substrate 10 is a glasssubstrate, it may be desirable to treat a surface of the glass substratewith an epoxy coating or a silane coating. For various polymer basematerials 10 it may be desirable to provide an intermediate layer 30 ofpolyamide, polyimide, polypropylene, polyethylene, orpoly(meth)acrylate. While not shown, it will be understood that SAcoating 20 may be disposed on a portion of intermediate layer 30. Itwill be further understood that intermediate layer 30 may be disposed ona portion of base material 10.

In various embodiments, surface 15 of base material 10 is treated,either physically or chemically, to impart a desirable property orcharacteristic to the surface 15. For example, and as discussed below,surface 15 may be corona treated or plasma treated. Examples of vacuumor atmospheric pressure plasma include radio frequency RF and microwaveplasmas both primary and secondary, dielectric barrier discharge, andcorona discharge generated in molecular or mixed gases including air,oxygen, nitrogen, argon, carbon dioxide, nitrous oxide, or water vapor.

SA coating layer 20, whether disposed on an intermediate layer 30 orbase material 10, preferably uniformly coats the underlying substrate.By “uniformly coated”, it is meant that the layer 20 in a given area,for example a surface of a well of a culture plate, completely coats thearea at a thickness of about 5 nm or greater. While the thickness of auniformly coated surface may vary across the surface, there are no areasof the uniformly coated surfaces through which the underlying layer(either intermediate layer 30 or base material 10) is exposed. Cellresponses across non-uniform surfaces tend to be more variable than cellresponses across uniform surfaces.

SA polymer coating layer 20 may have any desirable thickness. In variousembodiments, the average thickness of the coating layer 20 is less thanabout 10 micrometers. For example, the average thickness may be lessthan about 5 micrometers, less than about 2 micrometers, less than about1 micrometers, less than about 0.5 micrometers, between about 50 nm andabout 300 nm, or about 0.1 micrometers.

The polymer material forming SA layer 20 may be cross-linked to anysuitable degree. Low degree of crosslinking may result in partial orcomplete SA layer dissolution and lower polymerization reactionefficiency. In various embodiments, the crosslinking density of SA layer20 is between about 0.9% and about 9%.

Article 100, in numerous embodiments, is cell culture ware having asurface suitable for cell culture, such as a 6, 12, 96, 384, and 1536well plates, jars, petri dishes, flasks, multi-layered flasks, beakers,plates, roller bottles, slides, such as chambered and multichamberedculture slides, tubes, cover slips, bags, membranes, hollow fibers,beads and microcarriers, cups, spinner bottles, perfusion chambers,bioreactors, CellSTACK® and fermenters. In numberous embodiments thesecell culture surfaces are contained in a cell culture well. Referringnow to FIG. 2, article 100 formed from substrate or base material 10 mayinclude one or more wells 50. Well 50 includes a sidewall 55 and asurface 15. Referring to FIG. 2B-C, a SA coating 20 may be disposed onsurface 15 or sidewalls 55 (or, as discussed above with regard to FIG. 1one or more intermediate layer 30 may be disposed between surface 15 orsidewall 55 and SA coating 20) or a portion thereof. As shown in FIG.2C, 55 may be coated with SA layer 20.

In various embodiments, article 100 includes a uniformly coated layer 20having a surface 25 with an area greater than about 5 mm². Of course,the surface 25 may be of any suitable size. When the area of the surface15 is too small, reliable cell responses may not be readily observablebecause some cells, such as human embryonic stem cells, are seeded ascolonies or clusters of cells (e.g., having a diameter of about 0.5 mm)and adequate surface is desirable to ensure attachment of sufficientnumbers of colonies to produce a quantitative cell response. In numerousembodiments, an article 100 has a well 50 having a uniformly coatedsurface 15, where the surface 15 has an area greater than about 0.1 cm²,greater than about 0.3 cm², greater than about 0.9 cm², or greater thanabout 1 cm².

2. Coating of Synthetic Swellable (Meth)Acrylate Layer

A synthetic swellable (meth)acrylate (SA) layer may be disposed on asurface of a cell culture article via any known or future developedprocess. Preferably, the synthetic SA layer provides a uniform layerthat does not delaminate during typical cell culture conditions. Thatis, the SA layer is attached to the substrate or base material. Thesynthetic SA surface may be associated with or attached to the basematerial substrate via covalent or non-covalent interactions. Examplesof non-covalent interactions that may associate the synthetic SA surfacewith the substrate include chemical adsorption, hydrogen bonding,surface interpenetration, ionic bonding, van der Waals forces,hydrophobic interactions, dipole-dipole interactions, mechanicalinterlocking, and combinations thereof.

In various embodiments, the base material substrate surface is coatedaccording to the teachings of co-pending application Ser. No.12/362,782, filed on even date herewith, naming Zhou et al. asinventors, and entitled CELL CULTURE ARTICLE AND SCREENING, whichapplication is hereby incorporated herein by reference in its entiretyfor all purposes to the extent that it does not conflict with thedisclosure presented herein.

In numerous embodiments, monomers are deposited on a surface of a cellculture article and polymerized in situ. In such embodiments, the basematerial will be referred to herein as the “substrate” on which thesynthetic swellable (meth)acrylate material is deposited. Whilepolymerization may be done in solution phase or in bulk phase, inembodiments of the present invention, polymerization in bulk phase, insitu polymerization, yields a cell culture surface that is a networkedpolymeric surface. This networked polymeric surface is not aninterpenetrating polymeric network. In embodiments, the networkedpolymeric surface provided by in situ polymerization attaches to thesubstrate, does not delaminate, and survives in the aqueous environmentof cell culture without delaminating for periods of time that arerelevant for cell culture. In embodiments, the monomers are less than1000 daltons.

As monomers may be viscous, it may be desirable to dilute the monomersin a suitable solvent to reduce viscosity prior to being dispensed onthe surface. Reducing viscosity may allow for thinner and more uniformlayers of the synthetic swellable (meth)acrylate material to be formed.One of skill in the art will be able to readily select a suitablesolvent. Preferably the solvent is compatible with the material formingthe cell culture article and the monomers. It may be desirable to selecta solvent that is non-toxic to the cells to be cultured and that doesnot interfere with the polymerization reaction. Alternatively, or inaddition, selection of a solvent that can be substantially completelyremoved or removed to an extent that it is non-toxic or no longerinterferes with polymerization may be desirable. In such circumstances,it may be desirable that the solvent be readily removable without harshconditions, such as vacuum or extreme heat. Volatile solvents areexamples of such readily removable solvents.

Some solvents that may be suitable in various situations for coatingarticles as described herein include ethanol, isopropanol, acetylacetate, ethyl acetate, dimethylformamide (DMF), and dimethylsulfoxide(DMSO). As described in co-pending application Ser. No. 12/362,782,filed on even date herewith, entitled CELL CULTURE ARTICLE ANDSCREENING, volatile solvents, such as acetone, methanol, ethyl acetate,butanone, acetonitrile, isopropanol, and 2-butanol, and ethanol, may beparticularly suitable solvents when it is desired to remove solventprior to polymerization.

The monomers may be diluted with solvent by any suitable amount toachieve the desired viscosity and monomer concentration. Generally themonomer compositions used according to the teachings presented hereincontain between about 0.1% to about 99% monomer. By way of example, themonomer may be diluted with an ethanol or other solvent to provide acomposition having between about 0.1% and about 50% monomer, or fromabout 0.1% to about 10% monomer by volume, from about 0.1% to about 5%monomer by volume, or from about 0.1% to about 1% monomer by volume. Themonomers may be diluted with solvent so that the swellable(meth)acrylate layer achieves a desired thickness. As discussed above,if the deposited monomers are too thick, a non-uniform surface mayresult and the coating may likely de-laminate after contact with anaqueous medium.

In various embodiments, the synthetic swellable (meth)acrylate layer isdeposited on a surface of an intermediate layer that is associated withthe base material via covalent or non-covalent interactions, eitherdirectly or via one or more additional intermediate layers (not shown).In such embodiments, the intermediate layer will be referred to hereinas the “substrate” onto which the synthetic swellable (meth)acrylatelayer is deposited.

In various embodiments, the surface of the base material is treated. Thesurface may be treated to improve binding of the synthetic swellable(meth)acrylate layer to the base material surface, to facilitate monomerspreading on the base material surface, or the like. Of course, the basematerial may be treated for similar purposes with regard to anintermediate layer. In various embodiments, the surface is plasmatreated. High surface energy obtainable from such treatments mayfacilitate monomer spreading and uniform coating.

In addition to the monomers that form the swellable (meth)acrylatelayer, a composition forming the layer may include one or moreadditional compounds such as surfactants, wetting agents,photoinitiators, thermal initiators, catalysts, activators, andcross-linking agents.

Any suitable polymerization initiator may be employed. One of skill inthe art will readily be able to select a suitable initiator, e.g. aradical initiator or a cationic initiator, suitable for use with themonomers. In various embodiments, UV light is used to generate freeradical monomers to initiate chain polymerization.

Any suitable initiator may be used. Examples of polymerizationinitiators include organic peroxides, azo compounds, quinones, nitrosocompounds, acyl halides, hydrazones, mercapto compounds, pyryliumcompounds, imidazoles, chlorotriazines, benzoin, benzoin alkyl ethers,diketones, phenones, or mixtures thereof. Examples of suitablecommercially available, ultraviolet-activated and visiblelight-activated photoinitiators have tradenames such as IRGACURE 651,IRGACURE 184, IRGACURE 369, IRGACURE 819, DAROCUR 4265 and DAROCUR 1173commercially available from Ciba Specialty Chemicals, Tarrytown, N.Y.and LUCIRIN TPO and LUCIRIN TPO-L commercially available from BASF(Charlotte, N.C.)

A photosensitizer may also be included in a suitable initiator system.Representative photosensitizers have carbonyl groups or tertiary aminogroups or mixtures thereof. Photo sensitizers having a carbonyl groupsinclude benzophenone, acetophenone, benzil, benzaldehyde,o-chlorobenzaldehyde, xanthone, thioxanthone, 9,10-anthraquinone, andother aromatic ketones. Photosensitizers having tertiary amines includemethyldiethanolamine, ethyldiethanolamine, triethanolamine,phenylmethyl-ethanolamine, and dimethylaminoethylbenzoate. Commerciallyavailable photosensitizers include QUANTICURE ITX, QUANTICURE QTX,QUANTICURE PTX, QUANTICURE EPD from Biddle Sawyer Corp.

In general, the amount of photosensitizer or photointiator system mayvary from about 0.01 to 10% by weight.

Examples of cationic initiators include salts of onium cations, such asarylsulfonium salts, as well as organometallic salts such as ion arenesystems.

In various embodiments where the monomers are diluted in solvent beforebeing deposited on the substrate surface, the solvent is removed priorto polymerizing. The solvent may be removed by any suitable mechanism orprocess. In general about 80% or more, about 90% or more, about 95% ormore, or about 99% or more of the solvent may be removed prior topolymerization. As described in copending application Ser. No.12/362,782, filed on even date herewith, naming Zhou et al. asinventors, and entitled CELL CULTURE ARTICLE AND SCREENING, it has beenfound that removal of substantially all of the solvent prior to curing,allows for better control of curing kinetics and the amount of monomerconverted. When conversion rates of the monomers are increased, wastegeneration and cytotoxicity are reduced.

To form the synthetic swellable (meth)acrylate surface, the monomers arepolymerized. Whether polymerized in bulk phase (substantially solventfree) or solvent phase, the monomers are polymerized via an appropriateinitiation mechanism. Many of such mechanisms are well known in the art.For example, temperature may be increased to activate a thermalinitiator, photoinitiators may be activated by exposure to appropriatewavelength of light, or the like. According to numerous embodiments, themonomer or monomer mixture is cured using UV light. The curingpreferably occurs under inert gas protection, such as nitrogenprotection, to prevent oxygen inhibition. Suitable UV light combinedwith gas protection may increase polymer conversion, insure coatingintegrity and reduce cytotoxicity.

The cured synthetic swellable (meth)acrylate layer may be washed withsolvent one or more times to remove impurities such as unreactedmonomers or low molecular weight polymer species. In variousembodiments, the layer is washed with ethanol or an ethanol-watersolution, e.g. 70% ethanol, greater than 90% ethanol, greater than 95%ethanol or greater than about 99% ethanol. Washing with a 70% ethanolsolvent may not only serve to remove impurities, which may be cytotoxic,but also can serve to sterilize the surface prior to incubation withcells.

Swellable (meth)acrylate layers produced in accordance with theteachings herein form a single polymeric matrix layer. While such layersmay include local variations in structure and properties, the variationstend to be random, as opposed to interpenetrating networks described byothers. As discussed above, in many embodiments, a swellable(meth)acrylate layer as described herein is polymerized from monomers insitu while in contact with the substrate of the cell culture article.

A polypeptide may be conjugated to the polymerized swellable(meth)acrylate layer via any suitable technique. A polypeptide may beconjugated to a polymerized swellable (meth)acrylate layer 20 via anamino terminal amino acid, a carboxy terminal amino acid, or an internalamino acid. One suitable technique involves1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride(EDC)/N-hydroxysuccinimide (NHS) chemistry, as generally known in theart. EDC and NHS or N-hydroxysulfosuccinimide (sulfo-NHS) can react withcarboxyl groups of the swellable (meth)acrylate layer to produce aminereactive NHS esters. EDC reacts with a carboxyl group of the swellable(meth)acrylate layer to produce an amine-reactive O-acylisoureaintermediate that is susceptible to hydrolysis. The addition of NHS orsulfo-NHS stabilizes the amine-reactive O-acylisourea intermediate byconverting it to an amine reactive NHS or sulfo-NHS ester, allowing fora two step procedure. Following activation of the swellable(meth)acrylate layer, the polypeptide may then be added and the terminalamine of the polypeptide can react with the amine reactive ester to forma stable amide bond, thus conjugating the polypeptide to the swellable(meth)acrylate layer. When EDC/NHS chemistry is employed to conjugate apolypeptide to the swellable (meth)acrylate layer, the N-terminal aminoacid is preferably an amine containing amino acid such as lysine,ornithine, diaminobutyric acid, or diaminoproprionic acid. Of course,any acceptable nucleophile may be employed, such as hydroxylamines,hydrazines, hydroxyls, and the like.

EDC/NHS chemistry results in a zero length crosslinking of polypeptide70 to swellable (meth)acrylate layer 20. Linkers or spacers, such aspoly(ethylene glycol) linkers (e.g., available from Quanta BioDesign,Ltd.) with a terminal amine may be added to the N-terminal amino acid ofpolypeptide 70. When adding a linker to the N-terminal amino acid, thelinker is preferably a N-PG-amido-PEG_(X)-acid where PG is a protectinggroup such as the Fmoc group, the BOC group, the CBZ group or any othergroup amenable to peptide synthesis and X is 2, 4, 6, 8, 12, 24 or anyother discrete PEG which may be available.

In various embodiments, a 1 μM-2500 μM polypeptide fluid composition,such as a solution, suspension, or the like, is contacted with anactivated swellable (meth)acrylate layer to conjugate the polypeptide.For example the polypeptide concentration may be between about 100 μMand about 2000 μM, between about 500 μM and about 1500 μM, or about 1000μM. It will be understood that the volume of the polypeptide compositionand the concentration may be varied to achieve a desired density ofpolypeptide conjugated to the swellable (meth)acrylate layer.

The polypeptide may be cyclized or include a cyclic portion. Anysuitable method for forming cyclic polypeptide may be employed. Forexample and with reference to Example 4 below, an amide linkage createdby cyclizing the free amino functionality on an appropriate amino-acidside chain and a free carboxyl group of an appropriate amino acid sidechain. Also with reference to Example 4 below, a di-sulfide linkage maybe created between free sulfhydryl groups of side chains appropriateamino acids in the peptide sequence. Any suitable technique may beemployed to form cyclic polypeptides (or portions thereof). By way ofexample, methods described in, e.g., WO1989005150 may be employed toform cyclic polypeptides. Head-to-tail cyclic polypeptides, where thepolypeptides have an amide bond between the carboxy terminus and theamino terminus may be employed. An alternative to the disulfide bondwould be a diselenide bond using two selenocysteines or mixedselenide/sulfide bond, e.g., as described in Koide et al, 1993, Chem.Pharm. Bull. 41(3):502-6; Koide et al., 1993, Chem. Pharm. Bull.41(9):1596-1600; or Besse and Moroder, 1997, Journal of Peptide Science,vol. 3, 442-453.

3. Incubating Cells on Synthetic Swellable (Meth)Acrylate LayerContaining Conjugated Polypeptide

A cell culture article having a swellable (meth)acrylate layer withconjugated polypeptide as described above may be seeded with cells. Thecells may be of any cell type. For example, the cells may be connectivetissue cells, epithelial cells, endothelial cells, hepatocytes, skeletalor smooth muscle cells, heart muscle cells, intestinal cells, kidneycells, or cells from other organs, stem cells, islet cells, blood vesselcells, lymphocytes, cancer cells, primary cells, cell lines, or thelike. The cells may be mammalian cells, preferably human cells, but mayalso be non-mammalian cells such as bacterial, yeast, or plant cells.

In numerous embodiments, the cells are stem cells which, as generallyunderstood in the art, refer to cells that have the ability tocontinuously divide (self-renewal) and that are capable ofdifferentiating into a diverse range of specialized cells. In someembodiments, the stem cells are multipotent, totipotent, or pluripotentstem cells that may be isolated from an organ or tissue of a subject.Such cells are capable of giving rise to a fully differentiated ormature cell types. A stem cell may be a bone marrow-derived stem cell,autologous or otherwise, a neuronal stem cell, or an embryonic stemcell. A stem cell may be nestin positive. A stem cell, may be ahematopoeietic stem cell. A stem cell may be a multi-lineage cellderived from epithelial and adipose tissues, umbilical cord blood,liver, brain or other organ. In various embodiments, the stem cells arepluripotent stem cells, such as pluripotent embryonic stem cellsisolated from a mammal. Suitable mammals may include rodents such asmice or rats, primates including human and non-human primates. Invarious embodiments, the swellable (meth)acrylate-polypeptide layersupports undifferentiated culture of embryonic stem cells for 5 or morepassages, 7 or more passages, or 10 or more passages. Typically stemscells are passaged to a new surface after they reach about 75%confluency. The time for cells to reach 75% confluency is dependent onmedia, seeding density and other factors as know to those in the art. Ithas been found that stem cells cultured as described in the Examplesbelow reach about 75% confluency is between about 3 days and about 7days, generally being about 5 days.

Because human embryonic stem cells (hESC) have the ability to growncontinually in culture in an undifferentiated state, the hESC for use inthis invention may be obtained from an established cell line. Examplesof human embryonic stem cell lines that have been established include,but are not limited to, H1, H7, H9, H13 or H14 (available from WiCellestablished by the University of Wisconsin) (Thompson (1998) Science282:1145); hESBGN-01, hESBGN-02, hESBGN-03 (BresaGen, Inc., Athens,Ga.); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (from ES CellInternational, Inc., Singapore); HSF-1, HSF-6 (from University ofCalifornia at San Francisco); 13, 13.2, 13.3, 14, 16, 16.2, J 3, J 3.2(derived at the Technion-Israel Institute of Technology, Haifa, Israel);UCSF-1 and UCSF-2 (Genbacev et al., Fertil. Steril. 83(5):1517-29,2005); lines HUES 1-17 (Cowan et al., NEJM 350(13):1353-56, 2004); andline ACT-14 (Klimanskaya et al., Lancet, 365(9471):1636-41, 2005).Embryonic stem cells used in the invention may also be obtained directlyfrom primary embryonic tissue. Typically this is done using frozen invitro fertilized eggs at the blastocyst stage, which would otherwise bediscarded.

Other sources of pluripotent stem cells include induced primatepluripotent stem (iPS) cells. iPS cells refer to cells, obtained from ajuvenile or adult mammal, such as a human, that are geneticallymodified, e.g., by transfection with one or more appropriate vectors,such that they are reprogrammed to attain the phenotype of a pluripotentstem cell such as an hESC. Phenotypic traits attained by thesereprogrammed cells include morphology resembling stem cells isolatedfrom a blastocyst as well as surface antigen expression, gene expressionand telomerase activity resembling blastocyst derived embryonic stemcells. The iPS cells typically have the ability to differentiate into atleast one cell type from each of the primary germ layers: ectoderm,endoderm and mesoderm. The iPS cells, like hESC, also form teratomaswhen injected into immuno-deficient mice, e.g., SCID mice. (Takahashi etal., (2007) Cell 131(5):861; Yu et al., (2007) Science 318:5858).

Prior to seeding cells, the cells may be harvested and suspended in asuitable medium, such as a growth medium in which the cells are to becultured once seeded onto the surface. For example, the cells may besuspended in and cultured in a serum-containing medium, a conditionedmedium, or a chemically-defined medium. As used herein,“chemically-defined medium” means cell culture media that contains nocomponents of unknown composition. Chemically defined cell culture mediamay, in various embodiments, contain no proteins, hydrosylates, orpeptides of unknown composition. In some embodiments, conditioned mediacontains polypeptides or proteins of known composition, such asrecombinant growth hormones. Because all components ofchemically-defined media have a known chemical structure, variability inculture conditions and thus variability in cell response can be reduced,increasing reproducibility. In addition, the possibility ofcontamination is reduced. Further, the ability to scale up is madeeasier due, at least in part, to the factors discussed above. Chemicallydefined cell culture media are commercially available from Invitrogen(Invitrogen Corporation, 1600 Faraday Avenue, PO Box 6482, Carlsbad,Calif. 92008) as STEM PRO, a fully serum- and feeder-free (SFM)specially formulated from the growth and expansion of embryonic stemcells, Xvivo (Lonza), and Stem Cell Technologies, Inc. as mTeSR™1maintenance media for human embryonic stem cells.

One or more growth or other factors may be added to the medium in whichcells are incubated with the synthetic swellable (meth)acrylate layerconjugated to polypeptide (SAP). The factors may facilitate cellularproliferation, adhesion, self-renewal, differentiation, or the like.Examples of factors that may be added to or included in the mediuminclude muscle morphogenic factor (MMP), vascular endothelium growthfactor (VEGF), interleukins, nerve growth factor (NGF), erythropoietin,platelet derived growth factor (PDGF), epidermal growth factor (EGF),activin A (ACT) such as activin A, hematopoietic growth factors,retinoic acid (RA), interferons, fibroblastic growth factors, such asbasic fibroblast growth factor (bFGF), bone morphogenetic protein (BMP),peptide growth factors, heparin binding growth factor (HBGF), hepatocytegrowth factor, tumor necrosis factors, insulin-like growth factors (IGF)I and II, transforming growth factors, such as transforming growthfactor-β1 (TGFβ1), and colony stimulating factors.

The cells may be seeded at any suitable concentration. Typically, thecells are seeded at about 10,000 cells/cm² of substrate to about 500,000cells/cm². For example, cells may be seeded at about 50,000 cells/cm² ofsubstrate to about 150,000 cells/cm². However, higher and lowerconcentrations may readily be used. The incubation time and conditions,such as temperature, CO₂ and O₂ levels, growth medium, and the like,will depend on the nature of the cells being cultured and can be readilymodified. The amount of time that the cells are incubated on the surfacemay vary depending on the cell response desired.

The cultured cells may be used for any suitable purpose, including (i)obtaining sufficient amounts of undifferentiated stem cells cultured ona synthetic surface in a chemically defined medium for use ininvestigational studies or for developing therapeutic uses, (ii) forinvestigational studies of the cells in culture, (iii) for developingtherapeutic uses, (iv) for therapeutic purposes, (v) for studying geneexpression, e.g. by creating cDNA libraries, and (vi) for studying drugand toxicity screening.

As described in more detail in the Examples below, it has been foundthat the undifferentiated state of stem cells, such as embryonic stemcells, can be maintained for 5, 7, or 10 or more passages on articleshaving an SA-P substrate. In various embodiments, 50% or more, 60% ormore, 70% or more, or 80% or more of the cells remain undifferentiatedafter each passage. One suitable way to determine whether cells areundifferentiated is to determine the presence of the OCT4 marker, e.g.as described in more detail below in the Examples. In variousembodiments, the undifferentiated stems cells culture on SA-P surfacesfor 5, 7, or 10 or more passages retain the ability to bedifferentiated.

In the following, non-limiting examples are presented, which describevarious embodiments of the articles and methods discussed above.

EXAMPLES Example 1 Coating Preparation

Swellable (meth)acrylate coating surfaces were prepared from UVpolymerizable monomers and include a hydrophilic monomer, a carboxylgroup containing monomer, and a crosslinking monomer. Table 1 shows thecombination of swellable (meth)acrylate monomers employed. As shown inTable 1, formulations SA1A and SA1 have the same monomer composition.They differ from each other in that SA1A is diluted in ethanol in a 0.1%concentration v/v while SA1 is diluted in ethanol in a 0.25% v/vconcentration or higher. The dilutions of monomers diluted in ethanolare shown in Table 3. Table 2 shows the chemical structures of themonomers used.

TABLE 1 Swellable (meth)acrylate formulations employed Carboxyl groupFormulation Hydrophilic Monomer containing Crosslinking No. (vol. %)monomer (vol. %) monomer (vol. %) SA1A or hydroxyethyl 2-carboxyethylTetra(ethylene SA1 methacrylate (80) acrylate (20) glycol)dimethacrylate (3) SA3 hydroxyethyl 2-carboxyethyl Tetra(ethylenemethacrylate (60) acrylate (40) glycol) dimethacrylate (3) SA5poly(ethylene 2-carboxyethyl glycol)(600) acrylate (20) dimethacrylate(80) SA11 hydroxyethyl 2-carboxyethyl Tetra(ethylene methacrylate (90)acrylate (10) glycol) dimethacrylate (3) SA12 hydroxyethyl2-carboxyethyl Tetra(ethylene methacrylate (70) acrylate (30) glycol)dimethacrylate (3) SA13 Hydroxypropyl 2-carboxyethyl Tetra(ethylenemethacrylate(80) acrylate (20) glycol) dimethacrylate (3) SA142-Hydroxyethyl 2-carboxyethyl Tetra(ethylene acrylate(80) acrylate (20)glycol) dimethacrylate (3) SA15 1-vinyl-2- 2-carboxyethyl Tetra(ethylenepyrrolidone(80) acrylate (20) glycol) dimethacrylate (3) SA16Hydroxyethyl 2-Carboxyethyl Tetra(ethylene methacrylate (80) acrylate(20) glycol) dimethacrylate (1) SA17 Hydroxyethyl 2-CarboxyethylTetra(ethylene methacrylate (80) acrylate (20) glycol) dimethacrylate.(10) SA18 Di(ethylene glycol) ethyl 2-carboxyethyl Tetra(ethylene ethermethacrylate(80) acrylate (20) glycol) dimethacrylate (3) SA19Acrylamide(80) 2-carboxyethyl Tetra(ethylene acrylate (20) glycol)dimethacrylate (3) SA20 Ethylene glycol methyl 2-carboxyethylTetra(ethylene ether methacrylate(80) acrylate (20) glycol)dimethacrylate (3) SA21 3-sulfopropyldimethyl-3- 2-carboxyethylTetra(ethylene methacrylamidopropyl- acrylate (20) glycol) ammonium(80)dimethacrylate (3) SA22 Di(ethylene glycol) ethyl 2-CarboxyethylTetra(ethylene ether methacrylate(40) acrylate (20) glycol) Hydroxyethylmethacrylate(40) dimethacrylate. (3) SA23 Acrylamide (40) 2-CarboxyethylTetra(ethylene Hydroxyethyl methacrylate(40) acrylate (20) glycol)dimethacrylate. (3) SA24 Ethylene glycol methyl 2-CarboxyethylTetra(ethylene ether methacrylate (40) acrylate (20) glycol)2-Hydroxyethyl methacrylate (40) dimethacrylate. (3) SA253-sulfopropyldimethyl-3- 2-Carboxyethyl Tetra(ethylenemethacrylamidopropyl- acrylate (20) glycol) ammonium inner salt (40)dimethacrylate. (3) Hydroxyethyl methacrylate (40)

TABLE 2 Structures of Monomers 2-hydroxyethyl methacrylate

2-Carboxyethyl acrylate

Tetra(ethylene glycol) dimethacrylate

Tetra(ethylene glycol) diacrylate

poly(ethylene glycol)(600) dimethacrylate

Hydroxypropyl methacrylate

2-Hydroxyethyl acrylate

1-vinyl-2-pyrrolidone

Di(ethylene glycol) ethyl ether methacrylate

Acrylamide

Ethylene glycol methyl ether methacrylate

3-sulfopropyldimethyl-3- methacrylamidopropyl-ammonium

Isobutyl acrylate

Briefly, the monomers were diluted in ethanol to the concentration of(0.1%) or 0.25% by volume, and Durocur 1173 (3% volume/monomer volume)photoinitiator was added. The diluted monomer swellable (meth)acrylateformulations were added to a well of a plasma treated cyclic olefincopolymer 96 well plate (provided by Corning Life Science Developmentgroup) at a volume of 2 μL using BioTek Precision Pipetting System.

Each well received a predetermined swellable (meth)acrylate monomerformulation, with some wells being coated with MATRIGEL™ as a positivecontrol. The coatings were applied to result in an average thickness of0.5 micrometers or less. Unless stated otherwise, the resultingswellable (meth)acrylate coatings had an average thickness of about 0.1micrometers. For the wells coated with swellable (meth)acrylate monomerformulations, the ethanol solvent was removed by evaporation at roomtemperature for 3 hours in a fume hood. The coatings were then curedwith 13 mW/cm² pulsed (100 Hz) UV light (Xenon RC-801) for 1 min in N₂purged box (with fused silica window).

For a number of the swellable (meth)acrylate formulations, a well of a96-well plate was coated with 5 microliters of 0.1v/v % ethanol solutionof acrylic monomers or 2 microliters of 0.25 v/v % ethanol solution ofacrylic monomers, resulting in 5 nanoliters of monomers per well (0.3165cm²) and a coating thickness of 0.13-0.20 micrometers. The variousformulations and concentrations and volumes employed are listed in Table3 below.

TABLE 3 Swellable (meth)acrylate formulations and concentrations/volumesapplied Formulation No. 0.1% at 5 microliters 0.25% at 2 microlitersSA1A X SA1 X SA3 X SA5 X SA11 X SA12 X SA13 X SA14 X SA15 X SA16 X SA17X SA18 X SA19 X SA20 X SA21 X SA22 X SA23 X SA24 X SA25 X

Example 2 Polypeptide Conjugation to Swellable (Meth)Acrylate Surface

In a series of experiments designed to evaluate polypeptide conjugationto the swellable (meth)acrylate coatings prepared as described above, amixture of polypeptides (Ac-ArgGlyGlySerAspProlleTyrLys-NH₂ (SEQ IDNO:3)/Rhod-GlyArgGlyAspSerProIleIleLys-NH₂ (SEQ ID NO:4)) was conjugatedto a swellable (meth)acrylate coating of Formulation SA1A (see Tables 1,2 and 3) comprising 1-ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride (EDC)/N-hydroxysuccinimide (NHS) chemistry. Briefly, 50 μLof 0.1 mM EDC and 0.05 mM NHS solution in DMF were dispensed into a wellof 96-well cyclic olefin copolymer plate coated with swellable(meth)acrylate formulation. The activation of carboxyl groups wasallowed to proceed for 1-1.5 h, and then the activating solution wasaspirated. Immediately after that, 50 μL of polypeptide solution in 25mM phosphate buffer pH 7.4 were dispensed into the well and the reactionbetween well surface NHS esters and peptide primary amine groups wascarried out at ambient condition for 1.5 h and then the peptide solutionwas aspirated. Immediately after that, 50 μL of 1M ethanolamine, pH8.0-8.5 in water were dispensed into the well and the blocking reactionwas carried out at ambient condition for 1.5 h. Then, the blockingsolution was aspirated and the well was washed with phosphate buffer, 1%SDS solution, and finally deionized water. Fluorescence was quantifiedusing a Tecan microarray scanner. Alternatively, the polypeptides werequantified with the QuantiPro BCA assay kit available fromSigma-Aldrich. The peptide solution contained 1000 μM, 100 μM, 50 μM, 10μM, 5 μM, 1 μM, or 0 μM total peptide.

TABLE 4 Peptides conjugated to swellable (meth)acrylate surfaces andtested in hESC culture Peptide ID Peptide sequence (SEQ ID NO: 1) RGDYaa_(l)Xaa_(n)ArgGlyAspXaa_(m)Yaa_(l) (SEQ ID NO: 2) cyclicZaaXaa_(n)ArgGlyAspXaa_(m)Baa (SEQ ID NO: 3) (cyclic withYaa_(l)Xaa_(m)ZaaXaa_(n)ArgGlyAspXaa_(m)Baa conjugation Xaa_(m)Yaa_(l)sites) (SEQ ID NO: 4) BSP (mouse) LysGlyGlyAsnGlyGluProArgGlyAspThrTyrArgAlaTyr (SEQ ID NO: 5) BSP (mouse)AsnGlyGluProArgGlyAspThrTyrArgAlaTyr (SEQ ID NO: 6) cyclic BSPLysGlyGlyLys⁴AsnGlyGluProArgGlyAspThr TyrArgAlaTyrAsp¹⁷, Lys⁴-Asp¹⁷amide cycle (SEQ ID NO: 7) cyclic BSPLysGlyGlyLys⁴GluProArgGlyAspThrTyrArg Asp¹³, Lys⁴-Asp¹³ amide cycle (SEQID NO: 8) cyclic BSP LysGlyGlyCys⁴AsnGlyGluProArgGlyAspThr TyrArgAlaTyrCys¹⁷, Cys⁴-Cys¹⁷ disulfide bonded cycle (SEQ ID NO: 9) cyclic BSPLysGlyGlyCys⁴GluProArgGlyAspThrTyrArg Cys¹⁷ Cys⁴-Cys¹³ disulfide bondedcycle (SEQ ID NO: 10) BSP (mouse) XaaGlyGlyAsnGlyGluProArgGlyAspThrTyrArgAlaTyr (SEQ ID NO: 11) short FN (s- GlyArgGlyAspSerProLys FN)(human)(SEQ ID NO: 12) Long FN (l-FN) LysGlyGlyAlaValThrGlyArgGlyAspSerProAla(human) SerSer (SEQ ID NO: 13) CYR61 (mouseLysGlyGlyGlyGlnLysCysIleValGlnThrThrSer laminin) TrpSer GlnCysSerLysSer(SEQ ID NO: 14) Human LysTyrGlyLeuAlaLeuGluArgLysAspHisSerthrombospondin 1 Gly (SEQ ID NO: 15) SN (mouseLysGlyGlySerIleAsnAsnAsnArgTrpHisSerIle laminin) TyrIleThrArgPheGlyAsnMetGlySer (SEQ ID NO: 16) AG32 (mouseLysGlyGlyThrTrpTyrLysIleAlaPheGlnArgAsn laminin) ArgLys (SEQ ID NO: 17)C68 (mouse LysGlyGlyThrSerIleLysIleArgGlyThrTyrSer laminin) GluArg (SEQID NO: 18) C28 (mouse LysTyrGlyThrAspIleArgValThrLeuAsnArgLeu laminin)AsnThrPhe (SEQ ID NO: 19) C64 (mouseLysTyrGlySerGluThrThrValLysTyrIlePheArg laminin) LeuHisGlu (SEQ ID NO:20) A208 (IKVAV) LysTyrGlyAlaAlaSerIleLysValAlaValSerAla (mouse laminin)AspArg (SEQ ID NO: 21) C16 (mouseLysTyrGlyLysAlaPheAspIleThrTyrValArgLeu laminin) LysPhe (SEQ ID NO: 22)AG73 with LysTyrGlyArgLysArgLeuGlnValGlnLeuSerIle LysTyrGly ArgThrlinker (SEQ ID NO: 23) RNIA LysGlyGlyArgAsnIleAlaGluIleIleLysAspIle withlinker (SEQ ID NO: 24) AG-73 ArgLysArgLeuGlnValGlnLeuSerIleArgThr (SEQID NO: 25) RNIA peptide ArgAsnIleAlaGluIleIleLysAspIle (SEQ ID NO: 26)C16 (mouse LysAlaPheAspIleThrTyrValArgLeuLysPhe laminin) (SEQ ID NO: 27)Synthetic ArgGlyGlySerAspProIleTyrLys peptide (scrambled RGD) (SEQ IDNO: 28) Labeled RGD Rhod-GlyArgGlyAspSerProIleIleLys-NH₂ (SEQ ID NO: 29)VN (human) LysGlyGlyProGlnValThrArgGlyAspValPheThr MetPro (SEQ ID NO:30) RGE GlyArgGlyGluSerProIleTyrLys (SEQ ID NO: 31) BSP (modified)LysGlyGlyAsnGlyGluProArgGlyAspThrArg AlaTyr

Many of these sequences have a Lys-Gly-Gly (KGG) amino acid sequence atthe N-terminus. The Lys amino acid has a side chain containing an aminogroup and is used for conjugating the peptide to the activated(meth)acrylate coating. The Gly-Gly amino acids were used to serve as aspacer designed to project the putative RGD epitope away from the coatedsurface to enable optimal bio-specific interaction of the peptide withthe cell surface receptors. It is conceivable that the linker sequenceis not required or a shorter sequence can be used. Alternatively or inaddition, non-amino acid based linkers (such as poly ethylene oxidelinkers or the like) may be used as spacers (see, for example, FIG. 6).It will be understood that this sequence may be present or absent inembodiments of the peptide sequences of the present invention.

FIG. 3 shows fluorescence data regarding the ability of a mixture ofpeptides (Ac-ArgGlyGlySerAspProlleTyrLys-NH₂ (SEQ IDNO:27)/Rhod-GlyArgGlyAspSerProIleIleLys-NH₂ (SEQ ID NO:28)) to conjugateto a swellable (meth)acrylate coating of Formulation SA1A prepared asdescribed above. FIG. 3A is an image of the fluorescence intensity ofpeptide conjugated to average approximately 1 micrometer thick layers ofthe swellable (meth)acrylate coating, in increasing concentrations ofpeptide from 0 to 1000 μM. FIG. 3B is an image of the fluorescenceintensity of peptide conjugated to average approximately 0.1 micrometerthick layers of the swellable (meth)acrylate coating in increasingconcentrations of peptide from 0 to 1000 μM. FIG. 3C is a graph ofpeptide concentration (0 to 1 mM) vs. fluorescence intensity forpeptides coated on the 1 micrometer or 0.1 micrometer thick surfaces. Asshown in FIG. 3, the amount of peptide conjugated to the coatingincreases with peptide concentration in conjugation buffer. Fluorescenceintensities for the swellable (meth)acrylate coatings of twothicknesses, 1 μm and 0.1 μm, to which peptides were conjugated usingthe same protocol, suggest that peptides are immobilized at the surfaceof the coating, rather than evenly through the bulk of the swellable(meth)acrylate. If evenly through the bulk of the swellable(meth)acrylate, one would expect an order of magnitude difference influorescence intensities for these two coatings.

FIG. 4 shows the results of the QuantiPro BCA assay described in EXAMPLE2. Analyzing the peptide conjugated swellable (meth)acrylate resulted inan absorbance at 0.076 AU at 570 nm. Linearity was observed between 1and 15 μg/ml (as shown by the line in the figure). Although theresulting data was close to the limit of detection (1 μg/mL), theresulting density corresponded well to the density obtained fromfluorescence experiments (5.6 pmol/mm² for BCA and 8.6 pmol/mm² forfluorescence).

Example 3 HT-1080 Cell Adhesion and Proliferation Assays

To evaluate ability of a polypeptide conjugated to embodiments ofswellable (meth)acrylate using these conjugation methods to enable celladhesion and proliferation, peptidesAc-LysTyrGlyArgLysArgLeuGlnValGlnLeuSerlleArgThr-NH₂ (SEQ ID NO:22)(AG-73, an adhesive peptide) and Ac—Ac-GlyArgGlyGluSerProIleTyrLys-NH₂(SEQ ID NO:30) (RGE, a negative control) were conjugated to swellable(meth)acrylate (as described above) and adhesion of HT-1080 humanfibrosarcoma cells to the swellable (meth)acrylate was evaluated.Briefly, Laminin (5 μg/mL, Sigma-Aldrich) control wells were coated for1 hour at room temperature. All wells were blocked with 1% bovine serumalbumin (BSA) in phosphate buffered saline (PBS) for 1 hour at 37° C.Wells were washed briefly with PBS before incubation with 0.1% BSA inIscove's Modified Dulbecco's Medium (IMDM) prior to cell seeding.HT-1080 human fibrosarcoma cells (ATCC number: CCL-121) were grown inIMDM (Lonza) with 10% FBS (Lonza) to 90% confluency at standard cellculture conditions. Cells were trypsinized and allowed to recover inIMDM with 10% FBS for 30 minutes at 37° C., 5% CO₂. After recovery,cells were washed and resuspended in 0.1% BSA in IMDM and seeded onpeptide-conjugated plates at a density of 30,000 cells/well. Celladhesion was allowed to take place for 1 hour at standard cell cultureconditions. The media was aspirated from the wells and adherent cellswere fixed and stained in 50 μL of 0.2% crystal violet in 20% methanolfor 8 minutes at room temperature. Cellular absorption of crystal violetwas quantified through addition of 1% SDS in H₂O for 5 minutes prior toabsorbance measurement at 570 nm. For this assay, after polypeptideconjugation the remaining NHS esters on the swellable (meth)acrylatewere reacted with ethanolamine or aminopropyl morpholine to reduceuptake of crystal violet dye by the swellable (meth)acrylate.

FIG. 5 shows the results from the HT-1080 adhesion assay, showingadhesion of HT-1080 cells to embodiments of SAP surfaces of the presentinvention, as described in EXAMPLE 3. The relative number of adherentcells on swellable(meth)acrylate-peptide surfaces was normalized to thatof cells on Laminin (via OD570 nm). The peptides were tested at 100 μmAG-73 (SEQ ID NO:24), 10, 1 and 0 μM concentrations. OD570 nmmeasurements were taken, and the readings were normalized to Lamininadherence. “AG-73” is SAP-AG-73 (SEQ ID NO:24). “bAG-73” is SAP-AG-73(SEQ ID NO:24) which was blocked with ethanolamine prior to polypeptideconjugation. “RGE” is SAP-RGE (SEQ ID NO:30). RGE is a non-adhesivepeptide.

HT-1080 cells adhered specifically to adhesive peptideAc-LysTyrGlyArgLysArgLeuGlnValGlnLeuSerIleArgThr-NH₂ (SEQ ID NO:22)(AG-73) and did not adhere to surfaces (1) that were blocked withethanolamine prior to peptide conjugation (shown as bAG-73, or blockedAG-73) or (2) where a non-adhesive peptideAc-GlyArgGlyGluSerProIleTyrLy-NH₂ (SEQ ID NO:30) (RGE) was conjugated.100 μM peptide solution and 10 μM AG-73 peptide solution resulted in anadhesive surface, while 1 μM AG-73 peptide solution did not result incell adhesive surface. Very few cells adhered to SAP-bAG-73, indicatingthat AG-73 does not non-specifically adsorb to the synthetic polymericsurface.

Example 4 Assays for Culture of Undifferentiated Stem Cells

Cell adhesive peptides were selected for use with hESCs as listed inTable 4. This list of polypeptides contains peptides which have beenshown to target the two important cellular receptor classes of integrinsand heparan sulfate proteoglycans (HSPGs). These peptides also encompasssequences from the various domains and chains of laminin. Also, celladhesive peptides from other proteins have been included.

To screen the peptides shown in Table 4 for their ability to providesatisfactory cell culture surfaces for adhesive properties towardundifferentiated hESCs, peptides were conjugated to a swellable(meth)acrylate of formulation SA1A (see Tables 1, 2 and 3) using 1000 μMsolutions. Peptides were purchased from GenScript Corporation. Allpeptides were amidated at the C-terminus. To the N-terminus of selectedpeptides, spacers of repeating units of polyethylene glycol (PEG) wereadded. For example, PEO₄ refers to four repeating units of ethyleneglycol and PEO₁₂ refers to 12 repeating units of ethylene glycol. Thespacers contained a terminal amine. The spacers were added to thepolypeptides by GenScript Corporation.

Ac-LysGlyGlyAsnGlyGluProArgGlyAspThrTyrArgAlaTyr-NH₂ (SEQ ID NO:4) (theBSP sequence), was conjugated to the swellable (meth)acrylate coating ofFormulation SA1, SA1A and SA5 as described above in varyingconcentrations of peptide (1000 μM, 100 μM, 10 μM, 1 μM, and 0 μM).

All experimental plates were sterilized prior to the cell seeding byspraying with 70% ETOH, drying in a laminar hood, and washing five timeswith 200 μl Dulbecco's Phosphate Buffered Saline (DPBS). H7 hES cellswere seeded on peptide conjugated swellable (meth)acrylate (SAP)surfaces at a density of 35,000 cells/well (96-well plate) in 100 μl ofchemically defined medium Xvivo10 (Lonza, Cat #04-743Q) and supplementedwith 80 ng/ml basic fibroblast growth factor (bFGF) (R&D systems, Cat#234-FSE/CF) and 0.5 ng/ml transforming growth factor-β1 (TGFβ1) (R&Dsystems, Cat. #240B). MATRIGEL™ (MG)-coated wells were used as positivecontrol for adhesion of undifferentiated hES cells. Cells were culturedfor 48 hrs under standard cell culture conditions (37° C. with 5% CO₂)and then were fixed and processed for AttoPhos assay to measure alkalinephosphatase activity, which is a known marker for undifferentiated hEScells, and/or BCIP staining as described below.

Example 5 AttoPhos Screening

AttoPhos screening was performed as follows. Briefly, at the end ofincubation time, cells were rinsed with 150 μl of DPBS and fixed with 4%paraformaldehyde for 10 min at room temperature (70 μl/well of 96-wellplate). The cells were washed once with 150 μl of DPBS, and treated for10 min with 100 μl of AttoPhos fluorescent substrate for alkalinephosphatase (Promega) (diluted 1:3 in DPBS) protected from light.AttoPhos fluorescent intensity at 485/535 nm was obtained using Victor 3microplate reader (Perklin Elmer). AttoPhos fluorescent intensity forexperimental surfaces was expressed as % of MATRIGEL™ control.

Example 6 BCIP/NBT Staining

(5-Bromo-4-chloro-3-indolylphosphate (BCIP)/Nitroblue tetrazolium (NBT)staining of hES cells for colony morphology assessment was performed asfollows. Briefly, after obtaining AttoPhos fluorescent intensityreadings, cells were washed with 150 μl DPBS and processed for BCIPstaining to assess cell colony morphology. Seventy μl of BCIP/NBT wasadded to each well and incubated for 20-30 min. (to achieve desirablecolor intensity) at room temperature with a mild agitation. At the endof the staining, cells were washed once with 150 μl DPBS and eitherscanned or analyzed with light microscopy. H7 hES colony morphology onexperimental surfaces was compared to the colony morphology on MATRIGEL™(positive control).

FIG. 6 shows the results from the screening assay for peptide support ofundifferentiated stem cells using Formulation SA1A.Ac-KGGNGEPRGDTYRAY-NH₂ (SEQ ID NO:4), the BSP sequence demonstrated asignificant ability to support undifferentiated stem cells (56-60%AttoPhos fluorescence intensity (FI)) compared to MATRIGEL™ control.Some peptides were conjugated to PEO spacers/linkers (SEQ ID NO:25 andSEQ ID NO:26) as follows: NH₂PEO₄-ArgAsnIleAlaGluIleIleLysAspIle-NH₂(SEQ ID NO:25) or NH₂—PEO₄-LysAlaPheAspIleThrTyrValArgLeuLysPhe-NH₂ SEQID NO:26) where the PEO could be PEO₄ or PEO₁₂. As shown in FIG. 7,colonies of stem cells cultured on the surface containingAc-KGGNGEPRGDTYRAY-NH₂ (SEQ ID NO:4), the BSP sequence (FIG. 7B)demonstrated similar colony morphology to MATRIGEL™ (FIG. 7A) asassessed by BCIP staining of the cells. Growth of stem cells was notobserved on the swellable (meth)acrylate substrate (formulation no. 1)alone (FIG. 7C). FIG. 8 shows AttoPhos measurements, expressed as apercentage of MATRIGEL™ performance, for SA1 formulation, withincreasing peptide concentration at 1, 10, 100 and 1000 μM. As shown inFIG. 8, adhesion and growth of undifferentiated stem cells on swellable(meth)acrylate formulation SA1A surfaces containingAc-KGGNGEPRGDTYRAY-NH₂ (SEQ ID NO:4), the BSP sequence was peptideconcentration dependent. Together, these data suggest thatAc-KGGNGEPRGDTYRAY-NH₂ (SEQ ID NO:4), the BSP sequence, conjugated using1000 μM concentration, can support adhesion and growth ofundifferentiated H7 hES cells under chemically defined medium condition.

Referring now to FIG. 9, the way in which the swellable (meth)acrylatecoating is prepared and the components of the swellable (meth)acrylateaffect the ability of Ac-KGGNGEPRGDTYRAY-NH₂ (SEQ ID NO:4), the BSPsequence to support growth of undifferentiated stem cells. CB/TOP isuncoated cyclic olefin copolymer cell culture ware (plasma treatedTOPAS®). SA1A is a swellable (meth)acrylate formulation with and withoutconjugated Ac-KGGNGEPRGDTYRAY-NH₂ (SEQ ID NO:4) (as identified in FIG.6). SA5 an alternative swellable (meth)acrylate formulation shown inTable 1 with or without conjugated Ac-KGGNGEPRGDTYRAY-NH₂ (SEQ ID NO:4).MG is MATRIGEL™ control. As shown in FIG. 9, Formulation SA5 did notprovide a coating that supported undifferentiated hES cell culture inthe presence of chemically defined media comparable to that shown byMATRIGEL™. Without being limited by theory, this could be because theremay be a significant amount of poly(ethylene glycol) 600 withoutcarboxyethyl acrylate copolymerized at the ends, possibly creating abrush layer on the SA surface, instead of a network of polymers. Asshown in FIG. 9, Ac-KGGNGEPRGDTYRAY-NH₂ (SEQ ID NO:4), the BSP sequenceconjugated to Formulation SA1 resulted in comparable AttoPhos FI ascompared to MATRIGEL™ control. This is in contrast to Formulation SA1A,which showed about 60% of MATRIGEL™ AttoPhos FI with conjugatedAc-KGGNGEPRGDTYRAY-NH₂ (SEQ ID NO:4), the BSP sequence (as shown in FIG.6 and FIG. 8). As discussed above, the components of Formulations SA1and SA1A were essentially identical, with only the manner in which thecoating layers were prepared differing (SA1A: 0.1% at 5 microliters;SA1: 0.25% at 2 microliters). The thickness of the coating may beimportant. For example, in embodiments, it may be important to maintainthe SA coating thickness below 0.5 μm. Accordingly, the manner in whichthe swellable (meth)acrylate substrate is prepared, as well as thechemical composition and swellable (meth)acrylate structure can affectthe ability of a peptide-conjugated swellable (meth)acrylate surface tosupport culture of undifferentiated stem cells in a chemically definedmedium.

Example 7 Testing of Additional SA Coatings Conjugated toAc-KGGNGEPRGDTYRAY-NH₂ (SEQ ID NO:4), the BSP Sequence

Additional swellable (meth)acrylate layers (SA layers or coatings)conjugated to Ac-KGGNGEPRGDTYRAY-NH₂ (SEQ ID NO:4), the BSP sequencewere prepared and tested according to the EXAMPLES above. FIG. 10A andFIG. 10B are bar graphs of AttoPhos measurements of attachment andgrowth of undifferentiated H7 hES cells under chemically defined mediumcondition on different swellable (meth)acrylate formulations afterconjugated with peptide Ac-KGGNGEPRGDTYRAY-NH₂ (SEQ ID NO:4). TheAttoPhos fluorescent intensity (FI) for cells on all screened surfaceswas normalized to FI of cells on MATRIGEL™ (FIG. 10). All of theswellable (meth)acrylate formulations reported in FIG. 10A and FIG. 10Bdemonstrated similar or higher AttoPhos FI compared to MATRIGEL™ controland similar to MATRIGEL™ colony morphology as assessed by BCIP stainingof the cells (data not shown).

For the swellable (meth)acrylate formulations reported in FIG. 10, thenetwork was polymerized from either highly hydrophilic monomer such asacrylamide or mildly hydrophilic monomer such as hydroxypropylmethacrylate. The ratio of hydrophilic monomer ranged from 60 to 90%.The ratio of carboxyl-group containing functional monomer ranged from10% to 30%. The ratio of cross-linking monomers ranged from 1% to 10%.After conjugated with BSP-RGD peptide Ac-KGGNGEPRGDTYRAY-NH₂ (SEQ IDNO:4), all of the tested formulations supported similar or higher hESCattachment than the positive control, MATRIGEL™, in chemically defined,animal free condition.

Example 8 Testing of Additional Peptides Conjugated to SA1

In the EXAMPLES above, Ac-KGGNGEPRGDTYRAY-NH₂ (SEQ ID NO:4), the BSPpeptide, was shown to support culture of undifferentiated hESCs inchemically-defined, animal-free conditions when conjugated to aswellable (meth)acrylate polymer layer. Ac-KGGNGEPRGDTYRAY-NH₂ (SEQ IDNO:4), the BSP peptide incorporated the RGD adhesion epitope ofBonesialo protein (BSP) from mouse. Additional RGD containing peptideshave been identified. However, not all RGD sequences may result in thesame level of cell culture performance for every cell type. Some celltypes may culture better on different specific peptides. The sequencesflanking the RGD motif may significantly influence the degree of celladhesion and influence the post adhesion differentiation processes.Without being limited by theory, it may be that integrin mediated celladhesion may incorporate multiple cellular events of cell attachmentincluding cell spreading with stress fibers and focal adhesions. Withoutbeing limited by theory, it may be that peptide conjugated(meth)acrylate scaffolds allow for these integrin mediated cell bindingevents. In this Example, the impact of conjugating different RGDcontaining peptides to embodiments of the SA coating are explored.Specifically, data showing the response of undifferentiated stem cellsto embodiments of RGD peptides where the RGD epitope is derived from thehuman fibronectin protein conjugated to (meth)acrylate coatings arepresented.

Fibronectin is a cell adhesive protein. A polypeptide having thecell-attaching activity of fibronectin and incorporating the RGD epitopewas previously identified (see, e.g. U.S. Pat. No. 4,661,111). In thisEXAMPLE, a 15mer sequence (SEQ ID NO:12) (designated as long-FN or 1-FN)from the human fibronectin sequence was conjugated to the SA1 SAcoating. The impact of a shorter, commercially available (AmericanPeptide Co., Inc.) 7-mer fibronectin sequences (SEQ ID NO:11)(designated as short-FN or s-FN) conjugated to SA1 was alsoinvestigated. Without being limited by theory, it may be that shortpeptides are expected to have limited conformational stability and mayresult in reduced cell adhesion properties. The s-FN polypeptide is a6-mer sequence derived from human fibronectin where an additional Lysamino residue at the C-terminus. In addition to being half the size ofthe 15-mer epitope 1-FN, this 6-mer s-FN peptide will attach via theC-terminus and thus change the orientation of the RGD epitope on thesurface of the cell culture vessel.

In addition, due to the differences in the location of the conjugatingamino acid residue within the peptides the 1-FN and s-FN RGD sequencespresent the RGD epitope to extra-cellular integrins in two different andopposing orientations.

In this EXAMPLE, the 1-FN (SEQ ID NO:12), s-FN (SEQ ID NO:11) andAc-KGGNGEPRGDTYRAY-NH₂ (SEQ ID NO:4), the BSP peptides were conjugatedto a (meth)acrylate layer made from Formulation SA1 as described in theEXAMPLES above. AttoPhos studies were performed as described in EXAMPLE5. The results are shown in FIG. 11. FIG. 11 is a bar graph showingAttophos staining of hESCs grown on gradients of fibronectin conjugatedswellable (meth)acrylate. S—FN is the 7-mer fibronectin peptideconjugated acrylate. L-Fn is the 15-mer fibronectin peptide conjugatedacrylate. 10-, 100- and 1000-reflect the input peptide concentration(μM) for creating these coatings.

As shown in FIG. 11, the short fibronectin sequence (s-FN) is comparablein performance to the long fibronectin sequence, 1-FN. While the BSPsequence appeared to better support the culture of undifferentiated stemcells (relative to the fibronectin sequences), it may be that any aminoacid sequences of the form Yaa₁Xaa_(n)ArgGlyAspXaa_(m)Yaa₁ (SEQ ID NO:1)where Xaa and Yaa are any naturally occurring or synthetic amino acidand where at least one Xaa or Yaa has a functionality that enablesnucleophillic addition/amide bond formation with the surface carboxylgroups, would be acceptable choices for peptide based cell culturescaffolds.

Example 9 Testing of Additional Peptides Conjugated to SA1 CyclicPeptides

In the EXAMPLES above 15-mer Ac-KGGNGEPRGDTYRAY-NH₂ (SEQ ID NO:4), theBSP peptide was shown to support culture of undifferentiated hESCs inchemically-defined, animal-free conditions when conjugated to aswellable (meth)acrylate polymer layer. Without being limited by theory,it may be that short flexible peptides do not maintain their nativeconformation in aqueous media very well. The issue of conformationalinstability may be most prevalent in very short peptides. To mimic thepseudo-beta turn like conformation of the putative RGD epitope in thenative protein, strategies have been developed for peptide cyclizationusing very short peptides such as pentamers or octamers. In thisEXAMPLE, cyclic 13 and 17-mer RGD peptides are conjugated to embodimentsof SA coatings.

In addition, poly ethylene oxide (PEO) linker conjugated RGD sequencesare conjugated to embodiments of SA coatings. Without being limited bytheory, the addition of a spacer/linker may improve the bio-availabilityof the peptide epitope by projecting the peptide epitope away from theSA coating. In addition, the hydrophilic nature of the ethylene oxideunits in a PEO linker may reduce non-specific protein adsorption to thesurface. The linker was added to the polypeptide by American PeptideCompany in Vista, Calif.

All the peptides used in this EXAMPLE were synthesized at AmericanPeptide Company, Inc., 1271 Avenida Chelsea, Vista, Calif. 92081 or CEMCorporation, P.O. Box 200, 3100 Smith Farm Road, Matthews, N.C. 28106.

The following peptides were conjugated to a swellable (meth)acrylatelayer, Formulation SA1, as described in the EXAMPLES above. See Table 5for cyclic structures.

-   (i) Ac-LysGlyGlyAsnGlyGluProArgGlyAspThrTyrArgAlaTyr-NH₂ (SEQ ID    NO:4), “BSP”;-   (ii) NH2-PEO4-AsnGlyGluProArgGlyAspThrTyrArgAlaTyr-NH₂ (SEQ ID    NO:5), “linker BSP”;-   (iii)    Ac-LysGlyGlyLys4AsnGlyGluProArgGlyAspThrTyrArgAlaTyrAsp17-NH₂,Lys4-Asp17    amide cycle, (SEQ ID NO:6), “cyclic BSP 17-mer”;-   (iv) Ac-LysGlyGlyLys⁴GluProArgGlyAspThrTyrArgAsp¹³-NH₂, Lys⁴-Asp¹³    amide cycle, (SEQ ID NO:7), “cyclic BSP 13-mer”;-   (v) Ac-LysGlyGlyCys⁴AsnGlyGluProArgGlyAspThrTyrArgAlaTyrCys¹⁷-NH₂,    Cys⁴-Cys¹⁷ disulfide bonded cycle, (SEQ ID NO:8), “disulfide-BSP    17-mer” shown as FORMULA 4; and-   (vi) Ac-LysGlyGlyCys⁴GluProArgGlyAspThrTyrArgCys¹⁷-NH₂, Cys⁴-Cys¹³    disulfide bonded cycle, (SEQ ID NO:9), “disulfide-BSP 13-mer”.

TABLE 5 (SEQ ID NO: 6) Ac-LysGlyGlyLys⁴AsnGlyGluProArgGlyAspThrTyrArgAlaTyrAsp¹⁷-NH₂, Lys⁴-Asp¹⁷ “cyclic BSP 17-mer” amide cycle, (FIG.13)

(SEQ ID NO: 7) Ac- LysGlyGlyLys⁴GluProArgGlyAspThrTyrArgAsp¹³- NH₂,Lys⁴-Asp¹³ amide cycle “cyclic BSP 13- mer” (FIG. 13)

(SEQ ID NO: 8) Ac-LysGlyGlyCys⁴AsnGlyGluProArgGly AspThrTyrArgAlaTyrCys¹⁷-NH₂, Cys⁴-Cys¹⁷ disulfide bonded cycle, “disulfide-BSP17-mer” (FIG. 12)

(SEQ ID NO: 9) Ac-LysGlyGlyCys⁴GluProArgGlyAsp ThrTyrArgCys¹⁷-NH₂,Cys⁴-Cys¹³ disulfide bonded cycle, “disulfide-BSP 13-mer” (FIG. 12)

Two chemical strategies were utilized to synthesize cyclic peptides. Thefirst strategy utilized an amide linkage created by cyclizing the freeamino functionality on the lysine side chain and free carboxyfunctionality on the aspartic acid side chain. The second strategyutilized a di-sulfide linkage created between the free sulfhydryl sidechains on the two cysteine amino acids in the peptide sequence (seeWO1989005150). These cyclicizations were performed by CEM Corporation inMatthews, N.C.

The 13-mer and 17-mer amide linked cyclic peptide resulted in cyclicRGDs that were 10 and 14 amino acids, respectively. Similarly, the13-mer and 17-mer disulfide linked cyclic peptide resulted in cyclicRGDs that were 10 and 14 amino acids, respectively.

FIG. 12 shows adhesion of HT-1080 to SAP-BSP(cyclic) where the BSPpeptide was cyclized by disulfide bonds (SEQ ID NO:8) and (SEQ ID NO:9),Ac-LysGlyGlyAsnGlyGluProArgGlyAspThrTyrArgAlaTyr-NH2 (SEQ ID NO:4) (1000mM), and non adhesive peptide GRGESPIYK(SEQ ID NO:30) (RGE, 1000 mM)normalized to extracellular matrix protein fibronectin (FN, 5mg/mL)-coated surfaces. (SEQ ID NO:8) and (SEQ ID NO:9) cyclic BSPdisulfide peptide-conjugated (meth)acrylate surfaces supported HT-1080adhesion similar to Ac-LysGlyGlyAsnGlyGluProArgGlyAspThrTyrArgAlaTyr-NH2(SEQ ID NO:4)-conjugated (meth)acrylate. FIG. 13 shows that (SEQ IDNO:6) (amide cyclic BSP 17mer) and (SEQ ID NO:7) (amid cyclic BSP13mer), amide cyclized BSP peptide-conjugated (meth)acrylates alsosupported HT-1080 adhesion similarly to linear BSP-conjugated(meth)acrylates, Ac-LysGlyGlyAsnGlyGluProArgGly AspThrTyrArgAlaTyr-NH2(SEQ ID NO:4).

Results of AttoPhos staining of human embryonic stem cells (hESCs)cultured on a swellable (meth)acrylate layer with conjugated amidebonded, cyclized polypeptides are shown in FIG. 14. Briefly, H7 hESCwere cultured on SA conjugated with linear or cyclic BSP peptide to formSAP-BSP or SAP-BSP(cyclic) surfaces. BSP was cyclized using amidebonding. After 48 hrs in culture on SAP-BSP (SEQ ID NO:4) surfaces andSA coatings conjugated to (SEQ ID NO:6) (amide cyclic BSP 17mer) and(SEQ ID NO:7) (amid cyclic BSP 13mer), cells subjected to AttoPhosassay. MATRIGEL™-coated substrate wells were used as positive control.As shown in FIG. 14, H7 cells showed similar adhesion/growth response oncyclic and linear BSP conjugated SA surfaces. No significant differencein cell response was observed between 13mer and 17mer versions of amidebond-cyclized SAP-BSP(cyclic) surfaces. The results suggest that bothamide bonded, cyclized and liner BSP conjugated SA can supportadhesion/growth of undifferentiated H7 hESC.

FIG. 15 shows results of AttoPhos staining of human embryonic stem cells(hESCs) cultured on a swellable (meth)acrylate layer with conjugateddi-sulfide bonded, cyclized polypeptides (SEQ ID NO:8) (di-sulfidecyclic BSP 17mer) and (SEQ ID NO:9) (disulfide cyclic BSP 13mer)compared to SA-BSP (SEQ ID NO:4) and MATRIGEL™. Briefly, H7 hESC werecultured for 48 hours on SA conjugated with linear cyclic BSP peptidesto form SAP-BSP or SAP-BSP(cyclic) surfaces. After 48 hrs in culture,cells were fixed and subjected to AttoPhos assay. As shown in FIG. 15,H7 cells showed similar adhesion/growth response on cyclic and linearBSP conjugated SA surfaces. No difference in cell response was observedbetween 13mer and 17mer versions of SAP-BSP(cyclic) surfaces.Interestingly, disulfide bonded SAP-BSP surfaces demonstrated highercell AttoPhos response compared to linear SAP-BSP at lower (100 μM)concentration (compare AttoPhos FI for 100 uM peptide concentration inFIG. 15 and FIG. 8). The results suggest that, in embodiments, bothdisulfide bonded cyclic and liner BSP conjugated SA can supportundifferentiated H7 adhesion/growth, with cyclic BSP showing higher cellresponse at lower peptide concentration.

In embodiments the present invention provides a cell culture article,comprising: a substrate having a surface; a swellable (meth)acrylatelayer disposed on the surface of the substrate, wherein the swellable(meth)acrylate layer is formed from a composition comprising a carboxylgroup-containing (meth)acrylate monomer, a cross-linking (meth)acrylatemonomer, and a hydrophilic monomer capable of polymerizing with thecarboxyl group-containing (meth)acrylate monomer and the cross-linking(meth)acrylate monomer; and a peptide conjugated to a carboxyl groupresulting from the carboxyl group-containing monomer of the swellable(meth)acrylate layer. In embodiments, the swellable (meth)acrylate layerhas an equilibrium water content in water of between about 5% and about50% or between about 10% and about 40%. In embodiments, the carboxylgroup-containing (meth)acrylate monomer is 2-carboxyethylacrylate. Inembodiments, the cross-linking (meth)acrylate monomer is tetra(ethyleneglycol) dimethacrylate or tetra(ethylene glycol) diacrylate. Inembodiments the hydrophilic monomer is selected from the groupconsisting of hydroxyethyl methacrylate, hydroxypropyl methacrylate,2-hydroxyethyl acrylate, 1-vinyl-2-pyrrolidone, di(ethylene glycol)ethyl ether methacrylate, acrylamide, ethylene glycol methyl ethermethacrylate or 3-sulfopropyldimethyl-3-methacrylamidopropyl-ammonium.In embodiments, the ratio, by volume, of the cross-linking(meth)acrylate monomer to the carboxy group-containing meth(acrylate)monomer to the hydrophilic monomer is about 1-10 to about 10-40 to about60-90. Or, in embodiments, the ratio by volume, of the cross-linking(meth)acrylate monomer to the carboxy group-containing meth(acrylate)monomer to the hydrophilic monomer is about 3 to 20 to 80. Inembodiments, the swellable (meth)acrylate layer is free of polypeptidecrosslinkers.

In embodiments, the peptide conjugated to a carboxyl group resultingfrom the carboxyl group-containing monomer of the swellable(meth)acrylate layer comprises an amino acid sequence ofYaa₁Xaa_(n)ArgGlyAspXaa_(m)Yaa₁ (SEQ ID NO:1), where n is an integer of0 to 4, m is an integer of 0 to 5, 1 is 0 or 1 each Xaa is independentlyany native or biomimetic amino acid, and Yaa is independently any nativeor biomimetic amino acid With the proviso that peptide includes at leastone amino acid having functionality that enables nucleophilic additionto a free carboxyl group of the swellable (meth)acrylate layer, whereinthe free carboxyl group results from the carboxyl group-containing(meth)acrylate monomer. In embodiments, Yaa comprises lysine.

In additional embodiments, the peptide conjugated to a carboxyl groupresulting from the carboxyl group-containing monomer of the swellable(meth)acrylate layer comprises an amino acid sequence ofYaa₁Xaa_(n)ZaaXaa_(n)ArgGlyAspXaa_(m)Baa Xaa_(m)Yaa₁ (SEQ ID NO:3),where n is an integer of 0 to 4, m is an integer of 0 to 5, 1 is 0 or 1each Xaa is independently any native or biomimetic amino acid, Zaa andBaa are each independently any native or biomimetic amino acid havingcovalent bonds formed between atoms of their respective side chains toform a cyclic portion of the polypeptide, and Yaa is independently anynative or biomimetic amino acid; with the proviso that the polypeptideincludes at least one native or biomimetic amino acid havingfunctionality that enables nucleophilic addition to a free carboxylgroup of the swellable (meth)acrylate layer, wherein the free carboxylgroup results from the carboxyl group-containing (meth)acrylate monomer.In embodiments, Yaa comprises lysine.

In embodiments, the peptide conjugated to a carboxyl group resultingfrom the carboxyl group-containing monomer of the swellable(meth)acrylate layer comprisesLysGlyGlyAsnGlyGluProArgGlyAspThrTyrArgAlaTyr (SEQ ID NO:4);AsnGlyGluProArgGlyAspThrTyrArgAlaTyr (SEQ ID NO:5)LysGlyGlyLys⁴AsnGlyGluProArgGlyAspThrTyrArgAlaTyrAsp¹⁷ (SEQ ID NO:6),where Lys⁴ and Asp¹⁷ together form an amide bond to cyclize a portion ofthe polypeptide; LysGlyGlyLys⁴GluProArgGlyAspThrTryArgAsp¹³ (SEQ IDNO:7), here Lys⁴ and Asp¹³ together form an amide bond to cyclize aportion of the polypeptide;LysGlyGlyCys⁴AsnGlyGluProArgGlyAspThrTyrArgAlaTyrCys¹ (SEQ ID NO:8),where Cys⁴ and Cys'⁷ together form a disulfide bond to cyclize a portionof the polypeptide; LysGlyGlyCys⁴GluProArgGlyAspThrTryArgCys¹³ (SEQ IDNO:9), where Cys⁴ and Cys¹³ together form a disulfide bond to cyclize aportion of the polypeptide; GlyArgGlyAspSerProLys (SEQ ID NO:11); andLysGlyGlyAlaValThrGlyArgGlyAspSerProAlaSerSer (SEQ ID NO:12).

In embodiments, the swellable (meth)acrylate layer has a uniformthickness. In embodiments, the swellable (methacrylate) layer is lessthan about 2 micrometers in thickness.

Example 10 Doubling Time

H7 human embryonic stem cells, cultured as described in Example 4 above,were passaged every 4-5 days (around 75% confluency) using enzymaticsub-cultivation procedure (collagenase IV treatment), followed bywashing with DPBS, scraping and re-suspending in chemically definedculture medium. Cell colony morphology, cell number, viability andhESC-specific marker expression profile relative to cells cultured inparallel with Matrigel surface was assessed at each passage. Thedoubling time (DT) on each surface coating was assessed using thefollowing formula where DT=doubling time in hours; T=total time inculture in hours; D₀=seeding cell density and D=harvesting cell density:

DT=T×log 2/log D−log D ₀  FORMULA 2

FIG. 16 shows results of doubling time experiments for undifferentiatedH7 hESCs cultured under chemically-defined, animal produce free mediumconditions on a MATRIGEL™ (MG) surface, SA1 with conjugated definedRGD-containing peptide (BSP) (SEQ ID NO:4), and a SA1 conjugatedRGD-containing peptide with a linker (BSP-PE0₄)NH₂—PE0₄-AsnGlyGluProArgGlyAspThrTyrArgAlaTyr (SEQ ID NO:5) Briefly, H7hESC were cultured on SAP-BSP or BSP-PEO4 conjugated SA in 6-wll platesfor ten consecutive passages in chemically defined medium (Xvivo10+bFGFand TGFb1). Cells cultured on MATRIGEL™-coated substrate surface wereused as positive control. At each passage, cell doubling time, cellviability and expression of Oct4 hESC marker (data not shown) wasassessed. In embodiments, the culture of undifferentiated stem cellsthrough 10 passages is considered “long term culture” of these cells. Inembodiments, culture of cells for 5 passages, 7 passages, 10 passages,11 passages, 12 passages, 13 passages, 14 passages, 15 passages, 16passages, 17 passages, 18 passages, 19 passages, 20 passages or longeris considered long term culture. Those of skill in the art willrecognize that, although a population of cells in culture may be or maybecome mixed cell types, a sufficient percentage of cells in the cellculture must remain in the undifferentiated state for the passage to beconsidered successful. In embodiments of the present invention, passagesare successful passages which retain a sufficient percentage of cells intheir undifferentiated state so as to be considered an undifferentiatedpopulation of cells.

In embodiments A method for culturing an isolated population ofundifferentiated stem cells, comprising: providing a cell culturearticle having a substrate, a synthetic polymer layer disposed on thesubstrate, and a peptide conjugated to the synthetic polymer layer,wherein the peptide comprises and amino acid sequence of RGD; culturingthe undifferentiated stem cells on the polypeptide conjugated to thesynthetic polymer layer of the cell culture article in a chemicallydefined cell culture medium and maintaining the cells in culture for atleast 5 passages, at least 10 passages or more passages. In embodiments,the chemically defined culture medium comprises basic fibroblast growthfactor and transforming growth factor-β1. In embodiments, the stem cellsare human stem cells or human embryonic stem cells, which may be H1 orH7 human embryonic stem cells.

In additional embodiments include a method for culturing an isolatedpopulation of cells, comprising: providing a cell culture article havinga substrate, a swellable (meth)acrylate layer disposed on the substrate,and a peptide conjugated to the swellable (meth)acrylate layer, whereinswellable (meth)acrylate layer is formed from a composition comprising acarboxyl group-containing (meth)acrylate monomer, a cross-linking(meth)acrylate monomer, and a hydrophilic monomer capable ofpolymerizing with the carboxyl group-containing (meth)acrylate monomerand the cross-linking (meth)acrylate monomer, and wherein the peptidecomprises and amino acid sequence of RGD; contacting the cells with thepolypeptide conjugated to the swellable (meth)acrylate layer of the cellculture article; and culturing the cells in a chemically defined cellculture medium to maintain the cells in culture. In embodiments, thecells are undifferentiated stem cells, human embryonic stem cells, or H1or H7 cells. In embodiments, the method further comprises passaging thecells five or more times on additional cell culture articles inchemically the defined cell culture medium, wherein 50% or more of thecells remain in an undifferentiated state following each of the five ormore passages or ten or more times. In embodiments, the chemicallydefined culture medium comprises basic fibroblast growth factor andtransforming growth factor-β1 which may be .about 80 ng/ml basicfibroblast growth factor and about 0.5 ng/ml transforming growthfactor-β1.

As shown in FIG. 16, H7 hESC doubling time on SAP-BSP or SAP-BSP-PEO₄surfaces was more consistent compared to the doubling time of cellscultured on MATRIGEL™-coated surface, which fluctuated dramatically frompassage to passage. These fluctuations could be due to lot-to-lot andprep-to-prep inconsistency of MATRIGEL™-coated surface. Average celldoubling time on SAP-BSP and SAP-BSP-PEO₄ was 41(+/−6) hours, while onMATRIGEL™ average cell doubling time was 50(+/−12) his, reflectinghigher level of inconsistency on MATRIGEL™ surface.

Average cell viability and Oct4-positive cell fraction was identical onSAP-BSP and MATRIGEL™ surfaces: 86(+/−2) % for viability, 92(+/−4) % forOct4-positive cell fraction. The results suggest that, in embodiments,SAP-BSP and SAP-BSP-PEO₄ surfaces can support long term culture ofundifferentiated H7 hESC, including multiple passaging, withoutaffecting cell viability or undifferentiated status, but improvingconsistency of cell doubling time.

Example 11 Testing of Additional Cell Types

Human embryonic stem cells are difficult to culture. Therefore, they area good model to use to show the applicability of embodiments of cellculture surfaces to provide relevant cell culture conditions fordifficult-to-culture cells. However, many cell types are “anchoragedependent.” That is, many cells require extra cellular matrix or serumto remain healthy in long term cell culture. Without being limited bytheory, it may be that for adherent cells requiring serum containingmedia, the serum provides a variety of adhesion proteins in addition tomitogenic factors (such as various growth factors) needed for cellculture. Adhesion proteins (which may include fibronectin, laminin,collagen etc.) present in the serum may become absorbed on plastic cellculture ware to form attachment surfaces for adherent cells. ThisEXAMPLE shows that applications for embodiments of peptide conjugated(meth)acrylate coatings of the present invention are not limited to stemcells. The data reported in this EXAMPLE demonstrate that embodiments ofsynthetic peptide conjugated (meth)acrylate coatings of the presentinvention are capable of replacing the need for serum containing mediaor a biological coating.

In the following EXAMPLES, human adult mesenchymal stem cells (hMSC),cardiomyocytes (differentiated from H7 human embryonic stem cells),human fibrosarcoma (HT1080) cells and HepG2/C3A cells are tested againstembodiments of the peptide-conjugated swellable (meth)acrylate cellculture substrates of the present invention.

For the results presented in EXAMPLES 12-17, cells were cultured on asurface formed from Formulation SA1 of EXAMPLE 1 and EXAMPLE 2conjugated with Ac-LysGlyGlyAsnGlyGluProArgGly AspThrTyrArgAlaTyr-NH2(SEQ ID NO:4) (BSP) according to EXAMPLE 2. The cell culture surfaceswere prepared as described in EXAMPLE 1, unless indicated otherwise.

Example 12 Bone Marrow Derived hMSC Cells

Bone marrow derived hMSC (Lonza, cat #PT-2501) were expanded in 10% FBScontaining standard culture medium (MSCGM from Lonza, cat #PT-3001) on atissue culture treated T-75 flask for one passage. hMSC cells generallyrequire 2-10% serum in their culture media. On day 8, cells wereharvested at ˜80-90% confluency with trypsin, washed twice with DPBS andseeded at the density of 7,000 cells/cm² on either TCT-PS 6 well-platesform Corning or 6 well plates coated withAc-LysGlyGlyAsnGlyGluProArgGlyAspThrTyrArgAlaTyr-NH2 (SEQ IDNO:4)-conjugated swellable (meth)acrylate of formulation SA1. Bothsystems were studied with 3 different media conditions described below.

-   1) MSCGM, standard culture medium for hMSC culture supplemented with    10% fetal bovine serum (FBS) is the standard conditions used for    in-vitro culture of hMSCs. Used in conjunction with cell culture    treated culture ware.-   2) StemPro MSC-SFM (Invitrogen, cat #A10332-01), serum-free medium    designed for hMSC culture. These chemically defined media require    use with CellStart humanized ECM matrix (Invitrogen, cat #A10142).-   3) Xvivo10 (Lonza, cat #04-743Q)+GF (GF, growth factors −20 ng/ml    bFGF+0.5 ng/ml TGFb1)

Cells were re-fed with corresponding medium every 2-3 days. Celladhesion/spreading, morphology and growth was monitored every day. Onday 7 cells were harvested with trypsin and cell count was performedusing TB and hemacytometer. MSCGM supplemented with 10% FBS is thestandard media for in vitro culture of hMSC on plastic culture ware andwas the control for this study. StemPro MSC-SFM and the XVivo10+GF(Lonza) media were chosen as they are two well known chemically defined,serum free media used in the field. The StemPro MSC-SFM media isspecifically designed for the culture of hMSCs and contains proprietarygrowth factors etc. SAP-BSP surfaces were compared with these standardsusing both serum containing and chemically defined media.

FIG. 17A-C are photomicrographs showing hMSC growth on embodiments ofthe present invention compared to TCT, in three different mediaconditions. In FIG. 17A, MSCM+10% FBS media was used to culture hMSCs onSAP-BSP surfaces (upper images) and TCT (lower images). MSCM+10% FBSrepresents the standard media condition used for commercial culture ofhMSC for clinical applications. In the middle images, FIG. 17B, MSC+SFMis StemPro a specialized media marketed by Invitrogen Corp. for hMSCculture that contains proprietary cocktail of growth factors. In FIG.17C, Xvivo+GF was used. This media combination incorporates Xvivo basalmedia, supplemented with bFGF and TGFb1 as described above in EXAMPLE 4.

FIG. 17A shows that in 10% FBS-supplemented MSCM media, hMSC cellsappear similar on TCT surfaces and on embodiments of the SAP-BSPsurfaces of the present invention. FIGS. 17B and C show that in theabsence of FBS, SAP-BSP embodiments of the synthetic swellable(meth)acrylate surfaces of the present invention provide better cellgrowth than TCT surfaces. FIGS. 17 B and C show the morphology and cellnumber on all culture conditions on day 7. The cell number onBSP-acrylate surface is significantly higher than on TCT for bothserum-free conditions, with cell number in Xvivo+GF condition exceedingcell number in 10% FBS condition. In addition, initial cell attachmentand spreading (2-20 hrs) was more efficient in 10% FBS medium comparedto serum-free conditions for both surfaces. At later time points (3-7days) cell survival/proliferation on BSP-acrylate surface underserum-free conditions was higher than on TCT. Embodiments of the presentinvention can enable the culture of hMSCs in serum free media.

FIG. 18 is a bar graph showing a comparison of hMSC cell number on TCTand BSP-(meth)acrylate after 7 days in different defined mediaconditions, as defined above. While both surfaces perform comparably inthe presence of FBS, the SAP-BSP (BSP peptide acrylate) surface providesa significantly improved cell culture surface compared to TCT in theabsence of FBS.

Example 13 H7 hESC Cells

In EXAMPLES above, the use of embodiments of swellable (meth)acrylatesurfaces conjugated to peptides for in-vitro expansion of hESCs wasdescribed. In this EXAMPLE, additional data that demonstrates the use ofembodiments of SAP surfaces for long-term in vitro expansion of hESCcells. While data is shown for H7 hESCs, H1 hESC cell lines have alsobeen expanded on these surfaces with similar results. A representationof the comparison of doubling time for H7 cells on BSP conjugated andMatrigel coated surfaces in the media conditions presented in EXAMPLE 4is presented in FIG. 19. The data indicates that the synthetic SAPsurface is comparable or better in cell culture performance to Matrigel™coating. In addition, cells cultured on BSP conjugated acrylate surfaceshowed similar to MATRIGEL™ expression of hESC-specific markers asdetermined by flow cytometery analyses (FIG. 20).

Example 14 Flow Cytometry

At the end of each passage, H7 hES cells cultured on synthetic surfacesor MATRIGEL™ were analyzed for hESC marker expression using flowcytometry. The entire staining procedure was performed at 4° C. Briefly,for each sample 5×10⁵ cells (fixed and permibialized for Oct4, livecells for Tra-1-60, Tra-1-81, and SSEA4 markers) were re-suspended in 50microliter of blocking solution (10% HI goat serum in DPBS) andincubated for 15 min, followed by addition of 50 microliter ofmarker-specific primary antibody (0.5 microgram/sample) or correspondingisotype control (0.5 microgram/sample) in blocking buffer for 30 min.After washing with 2 ml Staining buffer (SB) from BD Biosciences), cellswere incubated with corresponding secondary antibody (0.25microgram/sample) in SB for 30 min protected from light. After washingwith SB, cells were stained with PI (2 microgram/ml in SB) for 5 min,for viability assessment. 30,000 gated events (gating was set forPI-negative viable cell population) were acquired for each sample usingthe FACS Calibur (BD Biosciences). All analyses were done usingCellQuest Pro software (BD). Results are shown in FIG. 20. Resultsshowed that expression of hESC-specific markers were very similar onMATRIGEL™ and on the SAP-BSP synthetic surface.

Example 15 Differentiation of H7 hESCs to Cardiomyocytes

The culture of undifferentiated hESC on embodiments of BSP conjugatedswellable (meth)acrylate was disclosed In EXAMPLE 13. In this EXAMPLE,the use of the same surface for the differentiation of these cells tocardiomyocytes is disclosed. Stem cell derived cardiomyocytes may beobtained by any suitable methods. One of ordinary skill would recognizethat this class of peptide conjugated (meth)acrylates can also be usedenable the differentiation of hESC cells into other lineages such aspancreatic islet cells, neural cells, osteogenic cells etc.

Laflamme et al., “Cardiomyocytes derived from human embryonic stem cellsin pro-survival factors enhance function of infracted rats, NatureBiotechnology, 25: 1015-1024 (2007) describes a method fordifferentiating hESCs in culture. Briefly, undifferentiated humanembryonic stem cells, such as those derived from the female H7 humanembryonic stem cell line, may be seeded on MATRIGEL™-coated plates at adensity of about 100,000 cells/cm² and re-fed daily with hES cell growthmedium (KO DMEM+20% Serum replacement, 1 mM 1-glutamine, 1% NEAA, 0.1 mM2-ME plus hbFGF at 80 ng/ml and TGFb1 at 0.5 ng/ml). To inducedifferentiation, KO-SR may be replaced with RPMI-B27 medium (availablefrom Invitrogen) supplemented with about 100 ng/ml human recombinantactivin A (available from R&D Systems) for about 24 hours, followed by10 ng/ml human recombinant BMP4 (available from R&D Systems) for 72hours. Of course, any other suitable method may be employed.

After long term culture of hESC on synthetic SAP-BSP surfaces inchemically defined medium, cells still retain ability to differentiateinto cell types of therapeutic interest. H7 cells were cultured onSAP-BSP surfaces for 10 passages and then were differentiated intocardiomyocytes (CM) using the same SAP-BSP surface. Directdifferentiation protocol of LaFlamme (Supra.) was used. Briefly, H7cells were first preconditioned for 10-14 days in “knock-outserum-replacement” media (KO-SR) [KO-DMEM (Invitrogen, #10829018); 20%KO-SR (Invitrogen #10828-028); 1% Non-essential Amino Acids (Invitrogen#11140-050); 1 mM L-Glutamine; 0.1 mM Beta-Mercaptoethanol; 80 ng/mlhbFGF and 0.5 ng/ml hTGF-131 (R&D Systems; #234-FSE/CF and #240-B). Uponformation of post-confluent cell monolayers, CM differentiation wasinduced by sequential addition of the following recombinant growthfactors in RPMI 1640 medium (Invitrogen #11875) supplemented with B27(Invitrogen # 17504044): human Activin A-100 ng/ml (R&D Systems#338-AC-CF) for 24 hrs followed by human BMP4 (bone morphongenic protein4)-10 ng/ml (R&D Systems #314-BP-CF) for 72 hrs. The cells were thenallowed to recover for an additional 2-week period in RPMI 1640-B27medium. Medium was exchanged every 2-3 days until beating CM wereobserved in cultures.

Example 16 Cardiomyocytes (differentiated from H7 Human Embryonic StemCells)

Based on information and belief, all published reports on hESCdifferentiation into physiologically relevant (functional)cardiomyocytes require substrates that are coated with Matrigel™ orother ECM proteins. The data reported in the present EXAMPLE demonstratethat embodiments of the SAP surfaces of the present invention, includingSAP-BSP synthetic RGD-containing polypeptide conjugated swellable(meth)acrylate surfaces are capable of replacing the need for 2D/3Dscaffolds coated with biological coatings.

For CM marker assessment, differentiated cells were either fixed on6-well plates and processed for Nkx2.5/α-actinin double immunostaining(shown on FIG. 21A) or harvested, fixed and processed for α-actinin(FIG. 21B) or Nkx2.5 (FIG. 21C) flow cytometeric analysis. These resultsshow that after long-term culture (10+passages) of undifferentiated H7hESC on SAP-BSP synthetic surface in chemically defined medium, cellscan still be differentiated into cardiomyocytes. The differentiationefficiency was very similar to that seen on MATRIGEL™ surface (data notshown). Also the data suggest that SAP-BSP surface can be used tosubstitute currently used MATRIGEL™ for direct differentiation of hESCinto cardiomyocytes.

Example 17 HT1080 Cells

Using HT1080 cells, we demonstrate in this EXAMPLE that this class ofsynthetic peptide conjugated (meth)acrylate coatings can providesuitable cell culture surfaces for any cell type that requiresbiological coatings or serum containing media.

The culture of human fibrosarcoma (HT 1080) cells were chosen todemonstrate the universality of the peptide conjugated acrylatesurfaces. HT 1080 cells are routinely grown in serum containing media.The ATCC recommended protocol for growth of this cell type requiresATCC-formulated Eagle's Minimum Essential Medium, (Catalog No. 30-2003)supplemented with 10% fetal bovine serum. It is believed that adhesionproteins within the serum facilitate the attachment of these cells tothe plastic surface.

For this EXAMPLE, HT-1080 (ATCC # CCL-121) cells were grown to 70%confluency in Iscove's Modified Dulbecco's Medium (IMDM, Lonza, 12-722Q)containing 10% fetal bovine serum (FBS, Lonza, 14-502F) in atissue-cultured treated T150 flask (TCT, Corning, 430825). Cell mediawas replenished at 70% confluency with either serum-containing (IMDM+10%FBS) or serum-free (IMDM) media for one day prior to cell harvest. Cellswere harvested at 100% confluency with 0.05% trypsin-EDTA(Gibco/Invitrogen, 25300) and washed twice with Dulbecco'sPhosphate-buffered saline (DPBS, Gibco/Invitrogen, 14190). Cells wereseeded at the density of 25,000 cells/cm̂2 in either serum containing orserum free IMDM (+16 ng/mL Recombinant Human FGF basic (R&D Systems234-FSE/CF) and +0.5 ng/mL Recombinant Human TGF-B1 (R&D Systems,240-B-002)) onto the following 96 well plates: TCT-PolyStyrene (TCT-PS,Corning, 3603), swellable (meth)acrylate (SA1) coated Xenor, and SAP-BSPcoated Xenor. Cells were re-fed with corresponding medium every 2-3days. Cell adhesion/spreading, morphology and growth was monitored everyday. A cell proliferation assay (CellTiter 96® AQueous One Solution CellProliferation Assay, Promega, G358B) was used to assess relative cellnumber on each surface at cell confluency—day 2 (serum containing) andday 5 (serum-free), according to the manufacturer's protocol. FIG. 22 isa bar graph comparing cell titer data of HT-1080-cells on TCT, SA1(meth)acrylate-coated and SAP-BSP conjugated coating, in the presence ofserum (S, black bar) and in the absence (SF, white bars) of serum. FIG.23 shows photomicrograph comparing the morphology of HT-1080 cells onTCT and (meth)acrylate coating and BSP conjugated coatings in thepresence and absence of serum. The data from the CellTiter assay isshown in FIG. 22 and a comparison of cell morphology is shown in FIG.23. TCT-PS is plasma treated TCT surface. SA1 is the SA1 formulationfrom Table 1. SAP-BSP is the BSP-peptide conjugated (SEQ ID NO: 4) SA1formulation. The first three images are serum-free conditions (SF, Day5) and the last panel is in the presence of serum, at day 2. This datashows the feasibility of using the RGD polypeptide conjugated swellable(meth)acrylate coatings for cell culture of HT-1080 cells. HT-1080 cellsprovide a representative of engineered cell lines that require the useof serum for attachment purposes.

Example 18 Attachment of HepG2/C3A Cells to SAP Layers with ConjugatedLaminin Polypeptides

HepG2/C3A cells (ATCC # CRL-10741) were plated at 25,000 cells per wellin 100 μl media in triplicate on SAP surfaces, BD BioCoat™ Collagen Iand Corning CellBind® treated Topas™ in a 96 well format and cultured atin Eagle's Minimum Essential Medium (ATCC # 30-2003) supplemented with1% penicillin-streptomycin (Invitrogen # 15140-155). The culture mediumwas not supplemented with fetal bovine serum (as recommended by the cellline supplier) nor was the SAP surface exposed to fetal bovine serum.The culture plates were incubated at 37° C., in 5% CO₂ and 95% relativehumidity for 24 hours. The cell culture plates having the SA surfaceswere prepared as described in EXAMPLE 1. Peptides were conjugatedaccording to EXAMPLE 2. The polypeptides conjugated to the SA layer arelisted in Table 5. Polypeptides exhibiting results better than 75%attachment compared to Collagen I (see FIG. 24) are marked with anasterix in Table 6. The line in FIG. 24 indicates 75% performance ofcells on Collagen 1.

TABLE 6 Polypeptides conjugated to SA layer Sequence Source CLS NoKGGGQKCIVQTTSWSQC Cyr61 res 224-240 *CLS 01005 SKS (SEQ ID NO: 13)KYGLALERKDHSG TSP1 res 87-96 CLS 01010 (SEQ ID NO: 14)KGGSINNNRWHSIYITRFG mLMα1 res 2179-2198 CSL 01015 NMGS (SEQ ID NO: 15)KGGTWYKIAFQRNRK mLMα1 res 2370-2381 *CLS 01020 (SEQ ID NO: 16)KGGTSIKIRGTYSER mLMγ1 res 650-261 CLS 01025 (SEQ ID NO: 17)KYGTDIRVTLNRLNTF mLMγ1 res 245-257 CLS 01030 (SEQ ID NO: 18)KYGSETTVKYIFRLHE mLMγ1 res 615-627 *CLS 01035 (SEQ ID NO: 19)KYGKAFDITYVRLKF mLMγ1 res 139-150 *CLS 01050 (SEQ ID NO: 21)KYGAASIKVAVSADR mLMα1 res2122-2132 CLS 01040 (SEQ ID NO: 20)Ac-CGGNGEPRGDTYRAY BSP CLS 01045 (SEQ ID NO: 4) Ac-CGGNGEPRGDTRAY BSP-YCLS 01080 (SEQ ID NO: 38) KYGRKRLQVQLSIRT mLMα1 res 2719-2730 CLS 01055(SEQ ID NO: 22) KGGRNIAEIIKDI LMβ1 CLS 01060 (SEQ ID NO: 23)Ac-KGGPQVTRGDVFTMP- VN CLS 01075 NH2 (SEQ ID NO: 39) GRGDSPK Short FNCLS 01065 (SEQ ID NO: 11) Ac-KGGAVTGRGDSPASS- Long FN CLS 01070 NH₂ (SEQID NO: 12)

The attachment of the HepG2/C3A cells was determined using aCytotoxicity Assay (Promega CytoTox 96® Non-Radioactive CytotoxicityAssay #G1780) which estimates the number of living cells attached to theculture surfaces. After 24 hours of in culture, the cells werevigorously washed with phosphate buffer, lysed via the introduction ofthe lysis buffer solution and incubation at 37 C for 1 hour to releaselactate dehydrogenase (LDH) enzyme from the viable cells that remainattached to the culture surfaces. The biological activity of thereleased lactate dehydrogenase enzyme was measured via absorbance at 490nm using a plate reader (Victor 3 from PerkinElmer).

The comparison of the estimated number of viable cells (absorbance ofLDH activity) on SAPs, Collagen I and CellBind® treated Topas™ is shownin FIG. 24. Comparison of the cell culture surfaces demonstrated thatCorning SAPs with laminin peptide sequences KGGGQKCIVQTTSWSQCSKS (SEQ IDNO:13), KGGTWYKIAFQRNRK (SEQ ID NO:16), KYGSETTVKYIFRLHE (SEQ ID NO:19),and KYGKAFDITYVRLKF (SEQ ID NO:22) support serum-free attachment ofHepG2/C3A cells≧75% of Collagen I and greater than CellBind® treatedTopas™. In addition, the HepG2/C3A cells cultured on the Corning SAPsexhibited similar cell morphology as the cells cultured on Collagen I.

Thus, embodiments of SYNTHETIC SURFACES FOR CULTURING CELLS INCHEMICALLY DEFINED MEDIA are disclosed. One skilled in the art willappreciate that the arrays, compositions, kits and methods describedherein can be practiced with embodiments other than those disclosed. Thedisclosed embodiments are presented for purposes of illustration and notlimitation.

1.-25. (canceled)
 26. A method of differentiating a pluripotent stemcell into a cardiomyocyte comprising culturing the pluripotent stem on asurface comprising a swellable (meth)acrylate layer conjugated to an RGDpeptide and contacting the pluripotent stem cells with a mediacomprising one or more growth factors.
 27. The method of claim 26,wherein the pluripotent stem cell is a human embryonic stem cell. 28.The method of claim 26, wherein the swellable (meth)acrylate layercomprises hydroxyethyl methacrylate.
 29. The method of claim 26, whereinthe swellable (meth)acrylate layer comprises 2-carboxyethyl acrylate.30. The method of claim 26, wherein the swellable (meth)acrylate layercomprises tetra(ethylene glycol) dimethacrylate.
 31. The method of claim26, wherein the swellable (meth)acrylate layer comprises hydroxyethylmethacrylate, 2-carboxyethyl acrylate, and tetra(ethylene glycol)dimethacrylate.
 32. The method of claim 26, wherein the growth factorsare activin and BMP4.
 33. The method of claim 32, wherein thepluripotent stem cells are contacted with activin first and then BMP4.34. The method of claim 26, wherein the cardiomyocytes express Nkx2.5.35. The method of claim 26, wherein the cardiomyocytes express actinin.36. The method of claim 26, wherein the RGD peptide is a fragment ofbone sialo protein.
 37. The method of claim 26, wherein the RGD peptideis a fragment of vitronectin.